Discrimination of osteoarthritic and rheumatoid human synovial cells in culture by nuclear image analysis.

Rheumatoid arthritic (RA) and osteoarthritic (OA) synovial cells in culture differ in their metabolic and proliferative behaviour. To assess links between these properties and nuclear changes, we used image analysis to study chromatin texture, together with nuclear morphometry and densitometry of OA and RA cells in primary culture. Chromatin pattern at the third day (D3) was heterogeneous and granular with chromatin clumps whereas at the final stage (D11) of culture a homogeneous and finely granular chromatin texture was observed. This evolution indicates global chromatin decondensation. These characteristics were more marked for RA than for OA nuclei. At each culture time, RA nuclei could be discriminated with high confidence from OA ones from parameters evaluating the organization of the chromatine texture. Nuclear image analysis is thus a useful tool for investigating synovial cell biology.

Methodological considerations on Feulgen-staining applied to cells in primoculture: the model of osteoarthritic synovial cells.

Human synovial cells in primoculture are an interesting model for the study of articular joint diseases and anti-rheumatic drugs. Based on results obtained by image cytometry of Feulgen-stained nuclei, we describe the heterogeneity of synovial cell populations and their progression during culture time in primoculture. Using the hydrolysis properties of the Feulgen reaction and their variations dependent on fixatives, we demonstrate the high acid-lability of the condensed chromatin observed in short term cultured nuclei compared to the acid-resistance of decondensed chromatin in long term cultured nuclei; these variations being probably induced by modifications in the molecular supra-organisation of chromatin during the aging of a culture. Finally, due to the cellular heterogeneity of the biological model and its evolution during culture progression, technical compromises are proposed to obtain optimal Feulgen staining, using Böhm-Sprenger fixative and a 1 h hydrolysis by 6 M HCl at 20 degrees C.

Combined effects of RU486 and tamoxifen on the growth and cell cycle phases of the MCF-7 cell line.

The antiproliferative effect of RU486 and its effect combined with tamoxifen on the growth and cell cycle kinetics parameters on the MCF-7 human carcinoma cells were investigated. When MCF-7 cells in the exponential growth phase were treated with RU486 (10(2) nmol/L), a time-dependent cell growth inhibition was observed which was significant 5 days after the beginning of treatment. This inhibition was accompanied by a time- and dose-dependent decrease in the percentage of S and G2-M phase cells and a concomitant increase in the percentage of cells in the G0/G1 phase of the cell cycle. With tamoxifen (10(5) pmol/L), growth inhibition was obtained after 4 days of treatment of cells, and the blockage of the cell cycle occurred in the G0/G1 phase. In the case of simultaneous treatment of MCF-7 cultures with RU486 (10(2) nmol/L) and tamoxifen (10(5) pmol/L), we observed a synergistic inhibitory effect on the proliferative rate for short treatment (less than or equal to 3 days), whereas RU486 or tamoxifen alone had no effect. For the intermediate treatment (4 days), the combined effect of RU486 and tamoxifen was not significant compared to the effect of tamoxifen alone. For the long treatment (greater than 4 days), there were no differences between the number of cells in the treated cultures under different experimental conditions, but all were inhibited compared to those in control cell cultures. This simultaneous treatment of cells does not induce any change in the distribution of cells in the different cell cycle phases compared to tamoxifen percentages. These results suggest that RU486 is a cell cycle phase-specific growth inhibitory agent, and a combination of antiestrogen/antiprogestin should be considered as a possible improvement in breast cancer endocrine therapy.

PIP: The antiproliferative effect of the antiprogestational agent RU-486 (100 nmol/L), alone and its additive effect in combination with the antiestrogenic tamoxifen (100,000 pmol/L) on the growth and cell cycle phases of the human breast cancer cell line MCF7, which is ER and PR positive, using image cytometry was investigated. On days 2, 3, 4, 5, 7, and 9, cells were harvested from control and RU-486-, tamoxifen, and RU-486- and tamoxifen-treated cultures and counted with an hemocytometer. Cell number increased in the RU-486-treated culture for the first 4 days as well as in the control culture. RU-486 treatment of cells for 5 days produced a significant time-dependent cell growth inhibition (P 0.01) compared with that in the control cells. With tamoxifen (100,000 pmol/L), growth inhibition was obtained after 4 days of treatment of cells, and the blockage of the cell cycle occurred in the G0.G1 phase. For the short treatment a significant synergistic growth inhibition (P 0.05 and P 0.01 on days 2 and 3, respectively) compared to RU-486 or tamoxifen alone, which had no effect compared to control cells. In the case of simultaneous treatment of MCF7 cultures with RU-486 (100 nmol/L) and tamoxifen (10000 pmol/L), a synergistic inhibitory effect of the proliferative rate was not produce an effect. During intermediate treatment (4 days), the combined effect of RU-486 and tamoxifen was not significant compared to the effect of tamoxifen alone. On the other hand, tamoxifen inhibited growth compared to that in control cells (P 0.05). These findings suggest that RU-486 is a cell cycle phase-specific growth inhibitory agent, and a combination of antiestrogen and antiprogestin agents may be of possible use in breast cancer endocrine therapy.

Effects of gossypol on the cell cycle phases in T-47D human breast cancer cells.

Since gossypol, a naturally occurring component of cottonseed oil, exhibits a broad spectrum of activities, we have examined it as an antitumor agent on breast cancer. The effects of different concentrations of gossypol on the T-47D human breast cancer cell cycle phases were studied using cytometric image processing on Feulgen stained nuclei. The proportion of cells at different cell cycle phases was determined by discriminate analysis of the image parameters and gave good classification ranging from 86 to 100%. Gossypol was found to increase the G0/G1 fraction of the T-47D cells. This cell kinetic alteration by gossypol was shown to be dose dependent and reversible. Complete reversal of the effect of gossypol was observed after four days with a simple change to gossypol-free medium. The cells then progressed into S and G2/M phase, thus indicating that gossypol-treated cells remain viable. Gossypol was shown to have a strong inhibitory effect on cellular proliferation in T-47D cells. It was also found that this agent is only toxic to cells at the highest dose tested (10 microM). The results of this study may be of clinical significance in the treatment of breast cancer, since gossypol shows strong antiproliferative properties.

Sex steroid hormone modulation of NADPH pathways in MCF-7 cells.

Hormonal modulation of glucose-6-phosphate dehydrogenase (G6PD) and of utilization pathways of NADPH generated by G6PD was studied in the MCF-7 human breast cancer cell line, using a quantitative cytochemical method. Our results show that G6PD is increased by 17 beta-estradiol (estradiol) and synthetic progestin (promegestone R5020). The synthetic antiestrogen tamoxifen has no effect on G6PD activity. When it is present in the medium with estradiol, tamoxifen can oppose the stimulatory effect of estradiol on G6PD activity. Mifepristone (RU 38486) has no effect on G6PD activity, but it inhibits the R5020 stimulation of G6PD activity. After MCF-7 pretreatment with estradiol, there is a much stronger stimulation of G6PD activity by R5020. When we studied the effect of the steroid on the two utilization pathways of NADPH generated by G6PD activity, we observed that, in the cells treated with estradiol, there is an increase in reducing equivalents generated by G6PD activity which only affects the NADPH2 pathway, and that there is cell growth stimulation. When tamoxifen is present in the medium, we found no effect on the NADPH utilization pathways, nor on cell growth. In the presence of R5020, the NADPH2 pathway activity is increased but, under our experimental conditions, there was no effect on cell growth. On the other hand, even though RU 38486 is without effect on total G6PD activity, it does cause a modification in the distribution of reducing equivalents: the NADPH2 pathway activity is decreased, while the NADPH1 pathway is stimulated.

Vitamin D3 and glucose-6-phosphate dehydrogenase in rat duodenal epithelial cells.

A microdensitometric method was employed to determine enzyme activities in situ in undisrupted tissue rat duodenum. The effect of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] on glucose-6-phosphate dehydrogenase (G6PD) activity and on the two utilization pathways of synthesized NADPH, H1 (mixed function oxidation) and H2 (biosynthesis), was studied. In normal animals, a crypt-to-villus gradient of G6PD activity and of both NADPH utilization pathways was observed. A high level of NADPH utilization occurred predominantly via the H2 pathway. In vitamin D-deficient rat animals, G6PD activity in the middle part of the villus was approximately 60% lower than in normal animals [10.05 +/- 0.35 vs. 3.95 +/- 0.26 (means +/- SE) A585.min-1.micron-3 X 10(5), P less than 0.001] with reduced NADPH utilization via the H2 pathway (8.39 +/- 0.49 vs. 2.73 +/- 0.43 A585.min-1.micron-3 X 10(5), P less than 0.001) but not the H1 pathway (1.65 +/- 0.17 vs. 1.22 +/- 0.19 A585.min-1.micron-3 X 10(5), P = NS). Intraperitoneal administration of 1,25(OH)2D3 (500 pmol) to vitamin D-deficient animals resulted in increased G6PD activity within 30 min (4.09 +/- 0.38 vs. 5.51 +/- 0.39 A585.min-1.micron-3 X 10(5), P less than 0.05), attaining normal levels within 2 h. The H2 but not the H1 pathway of NADPH utilization increased significantly in response to 1,25(OH)2D3. This increase is essentially located in the basal and middle parts of the villus. Thus 1,25(OH)2D3 may influence biosynthesis in the duodenum via stimulation of G6PD activity and the H2 pathway of NADPH utilization.

Comparative effects of 17 beta-estradiol, progestin R5020, tamoxifen and RU38486 on lactate dehydrogenase activity in MCF-7 human breast cancer cells.

The effects of 17 beta-estradiol (estradiol), synthetic progestin R5020 and their antagonists, tamoxifen (Tam) and synthetic RU38486 on lactate dehydrogenase (LDH) activity in MCF-7 human breast cancer cells during the growth period were studied. A specially developed quantitative cytochemical assay was used; LDH activity is expressed per cell, and is thus independent of the positive and negative growth effects of the hormones and antagonists. Estradiol and R5020 stimulated LDH activity after similar exposures (6-48 h) and the stimuli were concentration dependent over the range 10(-7) M to 10(-10) M. As for the antagonists, RU38486 stimulated LDH activity in much the same way as estradiol and R5020; Tam alone, on the other hand, does not stimulate LDH, but when added to estradiol, Tam inhibits estradiol mediated LDH activation. When present at half-stimulant concentration, estradiol + R5020 and estradiol + RU38486 exhibit additive effects on LDH activity. Thus LDH appears to be an interesting tool for the study of hormone and antagonist effects in MCF-7 breast cancer cells.

[Contribution of quantitative cytology and ploidy in the diagnosis and monitoring of tumors of the bladder. Study of 52 cases using the Samba analyser]. / Apport de la cytologie quantitative et de la ploïdie dans le diagnostic et la surveillance des tumeurs de la vessie. Etude de 52 cas par l'analyseur Samba.

Image processors and present specific software applications have now placed scanning microcytometry in the ranks of other conventional technics for routine laboratory examinations, particularly for urinary cytology and DNA measurements (ploidy). Conventional urinary sediment smears of 52 patients with bladder tumor were digitized, processed and statistically analyzed by the image analyzer, Samba. They were automatically classified and ploidy measurements were made and compared with 6 conventional cytological diagnostics for each smear. The Samba program assigns the cell images to two classes, negative and positive. 100% of the cells were correctly classed with respect to the cytologist's diagnostics. Within the positive class the degree of malignancy established by the program was the same as the cytologist's classifications. For smears of class III, the comparison of the diagnostics of the cytologists for each patient permitted the validation of the differential classification, negative-positive, made using quantitative cytology. The interpretation of the two DNA histogram parameters, the degree of ploidy and the proliferation index, provided an excellent prognosis of which patients would show tumoral recrudescence, as verified by follow ups.

Stimulatory effect of 1,25-dihydroxyvitamin D3 on the glucose-6-phosphate dehydrogenase activity in the MCF-7 human breast cancer cell line.

Human breast cancer cell lines have been shown to possess high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and their growth is inhibited by this steroid. The present study examines the effect of 1,25(OH)2D3 on the activity of glucose-6-phosphate dehydrogenase (G6PD) in cells of a human breast cancer cell line MCF-7. G6PD, an enzyme which controls the hexose monophosphate shunt, is elevated and sensitive to 17 beta-estradiol in breast tumors. G6PD activity was stimulated by 1,25(OH)2D3 in a dose-dependent manner at very low concentrations of steroid (10(-10)-10(-12) M). 1,25(OH)2D3 increased maximum velocity without modifying the affinity constant of the enzyme for glucose-6-phosphate.

Rapid (10-minute) stimulation of rat duodenal alkaline phosphatase activity by 1,25-dihydroxyvitamin D3.

Recent reports have suggested that the action of calcitriol is much more rapid than previously thought. It is thus possible that some actions do not depend on de novo protein synthesis. A precise microdensitometric technique has been used to characterize the time course of the intestinal brush border alkaline phosphatase (AP) response of rat duodenal villi to the administration of calcitriol as AP activity has been shown to be dependent on the vitamin D status of the animal. The technique enables AP activity to be determined in situ without tissue disruption. After ip administration of 200 ng calcitriol to vitamin D-deficient male Wistar AF rats, a biphasic AP response was observed with an early peak within 1 h (0.068 +/- 0.011 vs. 0.101 +/- 0.003 integrated extinction (IE) min.micron 3 X 10(3), P less than 0.05) and a second at between 6 and 8 h (0.088 +/- 0.005 vs. 0.172 +/- 0.003 IE/min.micron 3 X 10(3), P less than 0.001). In a further experiment, the early response to calcitriol was reexamined with observations at 0, 10, 30, 45, and 60 min after administration of either calcitriol or vehicle (n = 5 pairs per time point). AP activity was significantly increased in the calcitriol group compared with the vehicle-treated group as early as 10 min after administration (0.132 +/- 0.003 vs. 0.151 +/- 0.005 IE/min.micron 3 X 10(3), P less than 0.02) and reached a peak 45 min after administration at which time AP activity was equal to that found in normal vitamin D-replete animals (0.193 +/- 0.003 vs. 0.192 +/- 0.002 IE/min.micron (3) X 10(3), P greater than 0.5). The speed of this response indicates it to be unlikely to depend on de novo protein synthesis.

Effects of 17 beta-estradiol and R5020 on glucose-6-phosphate dehydrogenase activity in MCF-7 human breast cancer cells: a cytochemical assay.

Although increasing levels of glucose-6-phosphate dehydrogenase (G6PD) have been widely reported in human breast tumor tissue, the effects of 17 beta-estradiol and progesterone on this key enzyme of cellular growth processes have not been well documented. Cellular heterogeneity of breast tumor tissue, coupled with the low sensitivity of classical biochemical assays of G6PD, are the main sources of difficulties in these studies. In the present report, the effects of estradiol and a progesterone analogue (R5020) on G6PD activity were studied using the MCF-7 cell line and a cytochemical assay of G6PD activity. Our results show that: (a) this assay can be used to perform quantitative measurements of G6PD on individual cells using small number of cells (20-30); and (b) both estradiol (10(-8)-10(-5) M) and R5020 (10(-9)-10(-5) M) stimulate the G6PD activity in dividing MCF-7 cells. Maximal stimulation (20-30%) was obtained after 24 h of treatment. This combination of MCF-7 cells and cytochemical assay is suitable for further studies on the effects of estradiol and R5020 stimulation of G6PD activity in breast tumors.

Increased potassium permeability in erythrocytes from patients with hyperparathyroidism.

Parathyroid hormone (PTH) has been shown to modify Ca2+ and Na+ transport in several epithelia. The molecular mechanisms of these effects are poorly understood. We investigated here whether PTH may modify Na+ and K+ transport across the human red blood cell membrane in vitro and ex vivo. Fourteen patients with severe primary or secondary hyperparathyroidism and hypercalcemia were studied before and 5-7 days after surgical parathyroidectomy. Erythrocyte ouabain-sensitive as well as furosemide-sensitive Na+ efflux rates of the patients were comparable to that of healthy volunteers and remained unchanged after parathyroidectomy. Moreover, erythrocyte Na+ fluxes of control subjects remained unchanged when red blood cells were incubated in the presence of 1.0 IU/ml of bovine PTH (1-85). However, erythrocytes from hyperparathyroid patients showed a significant increase in passive K+ permeability when compared to that of healthy controls (p less than 0.05). This abnormality could be corrected in vivo after parathyroidectomy and in vitro using quinine, respectively. It is concluded that hyperparathyroidism induces a moderate increase in Ca2+ dependent K+ permeability of erythrocytes ("Gardos effect") which is reversible after parathyroidectomy.

Kinetic analysis of lactate dehydrogenase in cultured chondrocytes by quantitative cytochemistry.

Kinetic analysis of lactate dehydrogenase activity in intact cultured chondrocytes was performed in situ by coupling cell culture and microcytophotometry. Cells were cultured on glass microscope slides divided into eight chambers and studied during the growth cycle in monolayer areas. Lactate dehydrogenase activity was assayed by the reduction of neotetrazolium in the presence of phenazine methosulfate. Quantification of formazan deposits within the cells was performed by scanning and integrating microdensitometry at the isosbestic wavelength of 585 nm. Results indicate the following (a) A kinetic characterization was possible: apparent constants, Km and Ks of this two-substrate enzyme were graphically determined Ks = 1.05 +/- 0.08 and 0.56 +/- 0.05 mM for lactate and NAD respectively and Km = 0.64 +/- 0.03 and 0.37 +/- 0.02 mM for lactate and NAD respectively. (b) Inhibition by lactate concentrations above 10 mM and pyruvate concentration of 1 mM, is in agreement with the well known high anaerobic glycolytic metabolism of chondrocytes. This was confirmed by electrophoresis on cellulose acetate which demonstrated a M3-H isoenzyme form in cultured chick chondrocytes. This study shows that microcytophotometric analysis of lactate dehydrogenase in cultured chondrocytes may be an interesting alternative to mass culture cells followed by classical biochemical studies.

In situ biochemical studies of intestinal alkaline phosphatase in normal and phosphate-depleted rats by microdensitometry.

A quantitative microdensitometric study has been designed to characterize in situ intestinal brush border-bound alkaline phosphatase of rat duodenal villosities. Intestinal slices were incubated with beta-glycerophosphate as substrate. Free phosphate liberated was precipitated in presence of a lead reagent as lead sulfide. The precipitate was quantified in situ by scanning and integrating microdensitometry. Kinetic parameters of the reaction were determined at 37 degrees C, pH 8.8, in the middle part of the villosities. Apparent Michaelis constant (Km) for beta-glycerophosphate was found to be 8.16 +/- 0.56 mM (mean +/- S.E.). Maximal enzyme activation was obtained at pH 8.5. Maximal inhibition of enzyme activity was observed in the presence of L-phenylalanine (30 mM) or theophylline (5 mM). Along the villosity axis, enzyme activity rose from the crypt up to the midportion of the villosity and finally decreased at the tip region. In phosphate-depleted rats, enzyme activity was increased in all portions of the villosity, with conservation of the same activity gradient. In this situation, kinetic analysis showed a marked decrease of Km, i.e. 4.56 +/- 0.39 mM (mean +/- S.E.) as compared to normal rats.

Human parathyroid gland adenylate cyclase activity: inhibition by 24,25-dihydroxycholecalciferol in vitro.

1. Plasma membranes were prepared from parathyroid adenomas in patients with primary hyperparathyroidism and from hyperplastic glands obtained from patients with chronic renal insufficiency. The basal and isoproterenol- or sodium fluoride-stimulated adenylate cyclase activities were measured in membranes in the presence of several vitamin D3 metabolites. 2. 24,25-Dihydroxycholecalciferol (10 and 1000 pmol/l) decreased isoproterenol- and sodium fluoride-stimulated adenylate cyclase activities in membranes prepared from parathyroid glands. 1,25-Dihydroxycholecalciferol (1000 pmol/l) inhibited the isoproterenol-stimulated adenylate cyclase activity. 25-Hydroxycholecalciferol and vitamin D3 had no effect on adenylate cyclase activities. Basal adenylate cyclase activity was not affected by any of th vitamin D3 metabolites tested. 3. These results indicate that 24,25-dihydroxycholecalciferol inhibits the isoproterenol- and sodium fluoride-stimulated adenylate cyclase activities in parathyroid tissues. Such an inhibition could explain the very rapid decrease in parathyroid hormone secretion after 24,25-dihydroxycholecalciferol administration that has been previously reported.

[Demonstration of a microtubule protein (tubulin) in swine parathyroid cytosol]. / Mise en évidence d'une protéine microtubulaire (tubuline) dans le cytosol de la parathyroïde de porc

A cytosolic colchicine-binding protein has been studied in Porcine parathyroid glands. The molecular weight of this protein is 110,000 dalton with a 6 S sedimentation constant. Its colchicine affinity is 1.3 X 10(6) l/mole at 37 degrees C. Polymerization-depolymerization kinetics and vinblastin sulfate-induced precipitation are the same as that of a tubulin.