Biomedical subjects

J B Rognoni

Publications and source records attributed to J B Rognoni.

17 recordsLinked to original sources

PubMed1991

Combination of culture on collagen gels and glucose starvation for cloning human colon cancer cells. Obtention of clones exhibiting different patterns of enterocytic differentiation.

Glucose starvation has been widely used to select differentiated subpopulations from the heterogenous human colon cancer cell line HT29. We observed that the important cell loss elicited by culturing these cells in glucose-free medium could be limited when type I collagen gel was used as substratum instead of conventional plastic support. We took advantage of this property to develop a new protocol, which combined glucose starvation and culture on collagen gels, for cloning HT29 cells. Using this procedure we have isolated four clones that were characterized on the basis of morphological (optical and transmission electron microscopy), electrophysiological (determination of transepithelial electrical parameters) and biochemical (detection of villin, sucrase-isomaltase and carcinoembryonic antigen) criteria. These four clones expressed different patterns of enterocytic differentiation regarding to these criteria. These results confirmed the heterogeneity of the HT29 cell line. One of these clones, HT29-A7, which displayed numerous intercellular cysts that disappeared at confluency, appears as a complementary model in the study of epithelial biogenesis.

Cell Differentiation↗

PubMed1990

Impaired carcinoembryonic antigen release during the process of suramin-induced differentiation of the human colonic adenocarcinoma cell clone HT29-D4.

The establishment of a differentiated state of the human colic adenocarcinoma cell clone HT29-D4 can be obtained by two ways: 1) the removal of glucose and its replacement by galactose in the culture medium (Fantini et al.: Biology of the Cell 65:163-169, 1989); 2) the addition of suramin, a polyanionic compound, in the glucose-containing medium (Fantini et al.: Journal of Biological Chemistry 264:10282-10286, 1989). We investigated the release of CEA in the culture medium of glucose-deprived HT29-D4 cells (HT29-D4-Gal) and studied its alteration in suramin-treated HT29-D4 cells (HT29-D4-S). The amount of CEA released in the medium in function of time in culture of undifferentiated HT29-D4-Glu cells was very low (5 to 8 ng/10(6) cells/24 hours) and almost constant throughout the experiment whereas it increased sharply during differentiation of HT29-D4-Gal cells (380 ng/10(6) cells/24 hours after 9 days in culture). Surprisingly the amount of CEA released by differentiated HT29-D4-S cells remained very low and comparable with the one of HT29-D4-Glu cells. Moreover suramin, when added to CEA-producing HT29-D4-Gal cells, strongly inhibited its release. Radioiodination of cell surface proteins followed by immunoprecipitation using an anti-CEA monoclonal antibody showed the presence of a 180 kDa polypeptide, i.e., CEA, predominantly labeled in HT29-D4-Gal and -S cells. The total CEA cellular content was higher in HT29-D4-Glu and HT29-D4-S cells than in HT29-D4-Gal cells. When HT29-D4-Gal or -S cells were treated with the bacterial phosphatidylinositol phospholipase C (Pl-PLC) a similar level of CEA was released suggesting a similar type of CEA anchorage. The present data demonstrate that a decrease in CEA release (i.e., in HT29-D4-Glu and -S cells) corresponds to an increase in its overall cellular expression. These results are in favour of a regulatory mechanism, impaired by suramin, which determines the balance between the soluble and the membrane bound forms of CEA.

Adenocarcinoma↗

PubMed1990

Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose.

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.

Cell Division↗

PubMed1989

Suramin inhibits cell growth and glycolytic activity and triggers differentiation of human colic adenocarcinoma cell clone HT29-D4.

Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis inhibits growth factor-induced mitogenesis. In the present report, we show that suramin inhibits the growth of human colic adenocarcinoma cells HT29-D4 and rapidly induces their differentiation into enterocyte-like cells. As soon as 6 days after the addition of suramin (100 micrograms/ml) in the culture medium, the cells form a polarized monolayer of regular columnar cells with occluding junctions delimiting two distinct membrane domains (apical and basolateral) and an apical brush-border expressing alkaline phosphatase and sucrase-isomaltase. The process of differentiation is fully reversible when the drug is removed from the culture medium. We also show that suramin inhibits both glucose consumption and lactate production so that the glycolytic activity of the treated cells is lowered by 42%. This observation would shed some light on the complex mechanisms involved during the induction of HT29 cell differentiation when glucose is removed from the culture medium.

Adenocarcinoma↗

PubMed1989

Induction of polarized apical expression and vectorial release of carcinoembryonic antigen (CEA) during the process of differentiation of HT29-D4 cells.

HT29-D4 clonal cells can be induced to differentiate by a simple alteration of the culture medium, that is, by the replacement of glucose by galactose [Fantini, J., et al. (1986) J. Cell Sci., 83:235-249] as reported for the nonclonal HT29 cells [Pinto, M., (1982) Biol. Cell, 44:193-196]. An essential property of the HT29-D4 cell line is the fact that no cell loss occurs after the medium change, so that the differentiated cells can be considered as the true counterpart of the undifferentiated one. This model is particularly suitable to study morphological and biochemical events associated with the progressive establishment of the differentiation state. We report here that carcinoembryonic antigen (CEA), a 180 kDa glycoprotein originally described as a colon tumor associated antigen, is faintly expressed at the surface of undifferentiated HT29-D4 cells. These cells release a small amount of CEA (2.5 ng/10(6) cells/24 hr) in the culture medium. Fourty-eight hours after glucose substitution by galactose, both CEA cell surface expression and release are strongly enhanced as demonstrated by immunofluorescence and immunoprecipitation studies. Ten days after the medium change, the amount of CEA released reaches a maximum value of 130 ng/10(6) cells/24 hr, which remains stable for differentiated HT29-D4 cells cultured in glucose-free, galactose-containing medium (Gal-medium) for several months. HT29-D4 cells grown in Gal-medium in porous-bottom culture dishes generate leakproof epithelial monolayers. We have successfully performed an independent radioiodination of the apical and basolateral domains of these cells, followed by immunoprecipitation. We demonstrate that CEA is expressed exclusively at the apical surface of differentiated HT29-D4 cells, since the 180 kDa polypeptide was immunoprecipitated only when the radioiodination was performed at the apical side of the monolayer. Leakproof HT29-D4 monolayers cultured in permeable chambers were also used to demonstrate that CEA was exclusively released in the medium bathing the apical side of the cells. In conclusion, this study of cell surface CEA expression and CEA release during the process of differentiation of HT29-D4 cells demonstrated that 1) CEA cell surface expression and CEA release are correlated with cell differentiation; 2) CEA is expressed in the apical brush border membrane of differentiated HT29-D4 cells; and 3) CEA release is exclusively oriented toward the apical side of the polarized monolayer.

Adenocarcinoma↗

PubMed1989

Down regulation of hypertrophied follicular cell volume in thyroid hyperplastic gland.

In the present study, changes in thyroid follicular cell volume and its regulation have been investigated during the early involution of a hyperplastic goitre. Male Wistar rats were administered an iodine deficient diet for 6 months with propylthiouracil (PTU, 0.15%) during the last two months. At the end of iodine deficiency (day 0), some rats were killed and the others received a normal iodine diet. These rats were killed after different periods of iodine refeeding. Thyroid follicular cell volume was very high in hyperplastic gland whereas thyroid protein concentration was low. Thyroid follicular cell volume quickly decreased when rats were normally iodine refed, whereas thyroid protein concentration increased. Electron microscopal observations showed that thyroid follicular cells retained their endocrine aspect in hyperplastic state and throughout the iodine refeeding period. Using concomitant stereological and biochemical techniques, it is shown that the amount of cellular iodide and an unknown iodinated compound strongly increased during the early iodine refeeding. Plasma TSH was high on day 0 and remained at this level until day 8 whereas plasma T3 and T4 were low on day 0 and remained at this low level until day 4. The present data show that the involution of thyroid follicular cell volume is induced by iodide and mediated by an iodinated compound at least in the initial phase, and is independent of plasma TSH, T3, T4, so indicating the involvement of a thyroid autoregulatory mechanism. These changes in cell volume may be of importance in ion transport, i.e. in the metabolism of thyroid follicular cell during the early involution of the hyperplastic goitre.

Animals↗

PubMed1988

Monoiodotyrosine formation during thyroglobulin processing in Golgi vesicles.

A golgi-enriched subfraction was obtained from porcine thyroid glands by differential centrifugation. When incubated in a suitable medium, these vesicles were able to concentrate iodide from the medium and bind it to protein. The iodination process was inhibited by methylmercapto-imidazole and was increased by the addition of an H2O2 generating system to the medium. Analysis of the protein content of the vesicles revealed the presence of 18 and 12-13 S thyroglobulin molecules, lacking mannose residues, and containing only monoiodotyrosine. It is concluded that in vitro, iodination can begin before exocytosis, in the smooth-surfaced vesicles derived from the golgi apparatus, as soon as N-acetylglucosamine is incorporated onto the pre-thyroglobulin molecule.

Animals↗

PubMed1987

Involution of hyperplastic goitre in the adult male rat. Tissue compartment process with early iodide effect: a stereological and biochemical study.

The morphological and functional changes during involution of hyperplastic goitre have been investigated in the adult male rat. Male wistar rats received an iodine-deficient diet for 6 months and during the last 2 months received propylthiouracil (PTU, 0.15%). By the end of this treatment (day 0), a hyperplastic goitre was obtained. A normal iodine supply was then given and PTU withdrawn. During the first 8 days of iodine refeeding, the plasma thyrotropin (TSH) remained at a high level (ten times the control value), whereas the thyroid iodide content was low on day 0, markedly increased on day 1 and decreased on day 4. Plasma T3 and T4 levels remained unchanged for 4 days and only increased on day 8. The total thyroid protein concentration was low on day 0 and then increased rapidly on day 8 (by 34%). The volume density of colloid remained low and unchanged until day 8, when it started to increase. However, the thyroid epithelial cell volume and the volume density of capillaries were raised on day 0, decreased rapidly in the next 8 days and more slowly later on. The total number of thyroid epithelial cells was considerably raised in the hyperplastic gland. It did not vary until day 16, when it decreased slowly, reaching a plateau on day 45 above the control value. The present data show that involution of hyperplastic goitre in the rat is due essentially to a decrease in thyroid epithelial cell volume and to a reduction of the increased number of capillary blood vessels present. The decrease in the number of epithelial cells is only 16.5%, suggesting that the death of thyroid epithelial cells contributes little. Half the process of involution, which occurs from days 0 to 8, is controlled by the thyroid iodide concentration rather than TSH, indicating the involvement of a thyroid autoregulatory mechanism. It must be emphasized, however, that the discontinuous pattern of epithelial cell number during involution may indicate that some cells with larger nuclei and more rapid turnover disappear more quickly after iodine refeeding.

Animals↗

PubMed1987

Thyroid autoregulation: impact on thyroid structure and function in rats.

Global response of the thyroid to a 10-fold increase in plasma iodide concentration results only in modifications of basolateral transfer for iodide where, by a balance phenomenon between influx and efflux, a net thyroid intake of 1.2 micrograms I/day is constantly maintained. The other main steps of thyroid iodine metabolism, thyroglobulin (Tg) iodination, endocytosis, and hydrolysis, remain constant. A stationary hormone secretion delivery results. This constant state is not found at a cellular level, where structural changes are observed in correlation with functional regulation of the cell dynamic state. Thus, an increase in plasma iodide concentration results in an increase of the apical membrane area (40%), whereas the basolateral membrane area decreases (18%). The volume of the follicle lumen increases (76%). Nevertheless, neither epithelial cell volume nor the structure of microvilli are modified. Comparison of iodine fluxes through the apical and basolateral membrane of the epithelial cell shows that an increase in plasma iodide concentration is correlated to a decrease of the Tg iodination and endocytotic fluxes (45%). A regulation also appears for basolateral transfer of iodide, whereas the lysosome system does not modulate the hormone secretion mechanism.

Animals↗

PubMed1987

The modulation by 3,5,3'-triiodothyronine (T3) of pituitary T3 nuclear receptors in hypothyroid rats is inhibited by cycloheximide.

The density of T3 nuclear receptors is known to vary with tissues and physiopathological conditions, but the factors involved in their regulation are still unknown. We have previously shown in the anterior pituitary gland that T3 modulates its own receptors; the density of T3 receptors in hypothyroid rats is half that in normal rats, and one injection of T3 is able to restore normal density of T3 receptors within 1-3 h. To determine whether T3 has a direct action on the synthesis of its nuclear receptor, the effect of cycloheximide (Cy) on T3-induced nuclear receptor was studied. In addition, the relationship between the density of pituitary T3 receptors and the secretion of TSH in different thyroid states was examined. In normal rats one injection of Cy (0.5-8 mg/100 mg BW) induced within 3 h a dose-dependent reduction in the density of pituitary T3 receptors as well as an important decrease in plasma TSH, with no changes in T4, T3, or pituitary TSH content. In hypothyroid rats the 50% decrease in the density of pituitary T3 receptors was not further reduced by 1 mg Cy. However, when the same dose of Cy was given 30 min before T3 it completely inhibited the induction by T3 of its receptors. When Cy was given 30 min or 1 h after T3 the inhibition was only partial. An inverse correlation was found between the density of T3 receptors in the pituitary gland and plasma TSH (r = -0.8128) in all experimental groups except those treated with Cy; this drug had an inhibitory effect on both TSH secretion and the density of receptors. The present data, therefore, support the view that T3 in the pituitary gland may induce the synthesis of its own nuclear receptors and that the density of T3 receptors is also involved in the control of TSH secretion.

Animals↗

PubMed1987

Cell kinetics of thyroid epithelial cells during hyperplastic goitre involution.

Thyroid epithelial cell kinetics were investigated in rats when a normal iodine diet was re-established after a long period of iodine deficiency supplemented with propylthiouracil (0.15%) for the last 2 months. In the first (prelabelling) experiment, all rats were labelled with a single injection of [3H]thymidine 2 days before iodine refeeding in order to follow the fate of the prelabelled cells. In the second experiment, the pulse-labelling index at the time of killing was measured; for this purpose the rats received [3H]thymidine 1 h before death. In these two experiments autoradiography was performed on histological sections. Thyroids were excised on day 0 and then at various intervals up to day 73 of iodine refeeding. Plasma tri-iodothyronine (T3) and thyroxine (T4) were very low until day 4 and then increased to reach control values on day 30. Thyroid concentration of iodide rose to 20 times the value on day 0, remained at this high level until day 2, and then diminished on day 4 to reach the control value on day 16. Plasma TSH concentrations were very high in iodine-deficient rats and did not vary significantly until day 8, when they fell rapidly to reach the control value on day 30. Thyroid weight, raised on day 0, decreased relatively quickly until day 4, then more slowly until day 30. Total thyroid epithelial cell number, high on day 0 (30.7 x 10(6) cells) was constant until day 16, then decreased until day 30 at which time a plateau was reached.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Negative effect of iodide on the survival of newly divided epithelial cells in chronically stimulated rat thyroid.

The aim of this work was to investigate some aspects of the thyroid epithelial cell kinetics during the iodide-induced involution of a hyperplastic goitre in the rat. Rats were made iodine-deficient for 6 months, and propylthiouracil (PTU) (0.15%) was added to the diet during the last 2 months. Thereafter, rats were refed with iodide and PTU was removed (day 0). Forty-eight hours previously, all the rats were injected with tritiated thymidine ([3H]TdR) (1 microCi/g). Some animals were killed 1 hr or 24 hr after [3H]TdR injection (i.e. on day -2 and -1, day 0 corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following [3H]TdR injection. Autoradiography of thyroid sections, iodine determination of plasma iodide and protein-bound iodine (PBI), and RIA of plasma thyroid stimulatory hormone (TSH) were performed. Plasma TSH concentration was very high on day 0 of iodide refeeding (3000 +/- 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day 0, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day 0, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day 0, decreased relatively quickly until day 30, then more slowly until day 73. The [3H]TdR labelling index (LI) of the thyroid epithelial cells (TEC) was high on day 0 (56 +/- 3 labelled cells/10,000 cells), and 24 hr thereafter increased to 104 +/- 3, by division of the labelled cells. On day 1 of iodine refeeding, the LI had abruptly decreased to about half this value and then remained stable for 3 more days. Between day 4 and day 16, a progressive decline in the LI, (by about 3-4 per day), was observed. The LI showed no further modification, up to day 73, the longest period investigated. The decrease in LI occurred without any significant changes in the labelling intensity (grain count) of the remaining labelled cells between day 1 and 16, this indicates that no cell division took place during this period. The data are therefore interpreted as showing a biphasic elimination after iodide refeeding, of cells that were actively proliferating during the goitrous state.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Iodide accumulation into thyroid Golgi vesicles.

Pig thyroid Golgi vesicles incubated in a suitable medium were able to concentrate iodide from the medium. This trapping required the integrity of the vesicles, was time- and temperature-dependent, and was inhibited by a competitive inhibitor of iodide active transport (perchlorate), suggesting a facilitated transport mechanism.

Animals↗

PubMed1985

[Effect of cycloheximide on nuclear triiodothyronine receptors in the pituitary of the normal and hypothyroid rat].

Cycloheximide (Cy), an inhibitor of protein synthesis was found to provoke a dose-dependent decrease of the hypophysis T3 nuclear receptors (T3nR) concentration in normal rats. In thyroidectomized rats, the reduced T3nR density was found to be normalized within 3 hrs. after a single injection of T3. Pretreatment with Cy inhibited the T3 effect on its own receptors, whereas Cy given after T3 was partially or not effective. These data suggest that the half-life of T3nR in the hypophysis is short (about 3 hrs.), and that it depends on protein neosynthesis.

Animals↗

PubMed1985

[Possible regulation of the growth of thyroid tissue by plasma iodide].

Rats on iodine deficient diet for 6 months received propylthiouracil (PTU) (0.15%) during the last 2 months. At the end of this treatment, PTU was withdrawn and the rats were iodine refed. 48 hrs. before the iodine refeeding all rats were injected with 3H thymidine. The results showed that some prelabelled cells in the hyperplastic goitre preferentially disappeared during its involution and therefore are more sensitive to iodine.

Animals↗

PubMed1985

Thyroid morphological and functional heterogeneity: impact on iodine secretion.

Thyroid iodine turnover heterogeneity includes morphological (cellular and colloidal distribution space for iodide) and functional heterogeneity (hormone synthesis in the colloid). In 'normal' rats, both iodide actively trapped by the epithelial cell and that coming from deiodination of iodotyrosines present the same probability for thyroglobulin (Tg) iodination (Tg iodination flux: 4.0 +/- 0.3 micrograms I/day). A portion of the thyroid iodide is sequestered in the colloid lumen and is inoperative in the Tg iodination mechanisms. The masses of cell and colloid compartments are equivalent (0.018 +/- 0.002 micrograms I) while colloid iodide concentration is twice that of the cell (0.11 and 0.06, respectively). The turnover of about 3 micrograms I of colloid iodine (Tg) is follicle diameter-dependent (inter-follicular heterogeneity) and it is mainly characterized by 2 different half lives of 8 and 16 hours, respectively. Ninety percent of the thyroid iodine (hormone) secretion (1.10 +/- 0.11 micrograms I/day) is provided by this compartment rich in iodotyrosine residues (70%). The remaining 10% of iodine secretion is provided by a Tg pool (7 micrograms I) characterized by 2 compartments (intra-follicular heterogeneity) with slow and very slow turnovers. The longer the transit time of Tg molecules in the colloid, the higher their iodothyronine content.

Animals↗

PubMed1975

Age-dependent sensitivity of basal cells in mouse vaginal epithelium towards the diferentiating action of estrogen.

Following ovariectomy, the vaginal epithelium of the mouse is reduced to two layers of cells, the basal layer that constitutes the germinative compartment and surface layer. Under estrogenic influence, this tissue undergoes keratinization at the expense of asal cell only. The initially superficial cells are progressively sloughed, without taking part in the cornification process. We have shown previously that within the first 12 hours after estrogen administration (that is, before any change in mitotic activity is detected) an intermediate layer of cells is formed. Thurs, a migratory process is induced, which leads to a very rapid redistribution to basal cells into two layers...

Animals↗