Water permeability in human airway epithelium

Osmotic water permeability (P_f) was studied in spheroid-shaped human airway epithelia explants derived from nasal polyps by the use of a new improved tissue collection and isolation procedure. The fluid-filled spheroids were lined with a single cell layer with the ciliated apical cell membrane facing the outside. They were capable of surviving hours of experiment involving continuous superfusion of the bathing medium and changes of osmolarity. A new image analysis technique was developed for measuring the spheroid diameters, giving high time and measurement resolutions. The transepithelial P_f, determined by the changes of the apical solution osmolarity, was not influenced by the presence of glucose, Na⁺, or Na⁺/glucose-cotransport inhibitors in the bath, but was sensitive to the aquaporin (AQP) inhibitor HgCl₂. The measured P_f levels and the values of activation energy were in the range of those seen in AQP-associated water transport. Together, these results indicate the presence of an AQP in the apical membrane of the spheroids. Notably, identical values for P_f were found in CF and non-CF airway preparations, as was the case also for the calculated spontaneous fluid absorption rates.     

Skin and conjunctival biopsies in infantile neuroaxonal dystrophy

Summary The diagnosis of infantile neuro-axonal dystrophy (INAD) in a 5-year-old patient was confirmed by the ultrastructural study of neuromuscular, skin and conjunctival biopsy specimens. Abnormal networks of smooth membranous, lamellar and tubular profiles were found in presynaptic terminals and in conjunctival and dermal axons.INAD is the first neurological disease outside the group of storage disorders in which skin and conjunctival biopsies contribute significantly to the diagnosis.     

La dystrophie neuroaxonale infantile ou maladie de Seitelberger

Résumé Les auteurs rapportent 2 cas de DNAI.Le tableau clinique, d'évolution mortelle en 2 ans, associe un arrêt du développement psycho-moteur, des signes pyramidaux et extrapyramidaux, un nystagmus pendulaire avec atrophie optique. L'étude en microscopie optique définit:—les aspects morphologiques, la nature glycolipoprotéique et la répartition topographique des sphéroïdes, électivement disposés dans les structures médullaires, bulbo-protubérantielles et cérébelleuses, phylogénétiquement les plus anciennes, —l'atrophie du cortex cérébelleux, —la surcharge soudanophile avec état dysmyélinique du pallidum, —la dégénérescence des voies afférentes et efférentes des fibres longues de la moelle, du tronc cérébral et des voies optiques.L'étude en microscopie électronique précise la structure des sphéroïdes composés d'amas membrano-tubulaires et cisternaux, des corps denses amorphes ou lamellés, multigranuleux, pseudo-cristallins ou multivésiculaires et de mitochondries pathologiques. La formation de ce matériel est liée à l'hyperplasie du réticulum endoplasmique lisse et à l'accumulation de mitochondries anormales dans les péricaryons neuronaux, les prolongements dendro-axonaux, les culs de sacs synaptiques. Les auteurs rapprochent leurs constatations de faits semblables observés en pathologie humaine et expérimentale.

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Infantile neuroaxonal dystrophy or seitelberger's disease. clinical, histological and ultrastructural study of 2 observations

Summary Two cases of INAD are reported. Clinical features with fatal issue within 2 years, are characterized by psychomotor retardation, pyramidal and extrapyramidal signs, pendular nystagmus with bilateral atrophia of optic discs.Light microscopy shows:—the morphology of spheroids, their glycolipoproteid component and their elective distribution in medulla, brain-stem and cerebellum, phylogenetically the oldest parts of the CNS—a cerebellar cortical atrophia—sudanophilic fatty deposits associated to status dysmyelinatus of pallidum—a degeneration of pyramidal, spinocerebellar tracts and optic pathways.Electron microscopy in the two cortical biopsies allows to specify the internal structure of spheroids made of membrano-tubular and interconnected aggregates, amorphous and lamellar electron-dense bodies, multigranular bodies, cristalline-like inclusions, multivesicular bodies and involved mitochondria. That complex material represents an overproduction of smooth endoplasmic reticulum and abnormal mitochondria located in neuronal perikarya, axons, dendrites and enlarged synaptic bulbs. These pathological findings are compared to those already described in human and experimental cases.

     

Short exposure to millimolar concentrations of ethanol induces apoptotic cell death in multicellular HepG2 spheroids

Purpose: We have shown previously that 1 mM ethanol reduces cell proliferation and increases apoptosis in monolayers of human hepatocellular carcinoma (HepG2) cells. However, in vivo liver tumors are usually three-dimensional and multicellular. The purpose of this study was therefore to determine the effect of ethanol in multicellular tumor spheroids (MCTS) as a model system in vitro. Methods: After the application of 1 mM ethanol for 24 h and 48 h, viable, apoptotic and necrotic cells within MCTS were stained with specific fluorescent dyes, and their amount and distribution within the MCTS were assessed by confocal laser scanning microscopy. To evaluate the effect on HepG2 cell migration and cell proliferation, the outgrowth potential after 1 week in culture was evaluated. Results: As assessed by YO-PRO-1 staining, ethanol increased the number of apoptotic cells from 21.5 units (U) in control spheroids to 364 U and 482.2 U after 24 h and 48 h in ethanol-treated spheroids, respectively (P < 0.001). Merocyanine staining fluorescence increased from 10.7 U in the control to 122 U after 24 h and 293.2 U after 48 h (P < 0.001). Cell viability, as determined by staining with the acetoxymethyl ester of calcein, decreased from 578.5 U in the control to 236 U and 73.4 U after 24 h and 48 h of ethanol exposure respectively (P < 0.001). Necrosis showed an increase from 2 U in control to 24.9 after 24 h and 54 U after 48 h. MCTS treated with ethanol showed almost complete inhibition of outgrowth potential after 1 week in culture, compared to controls (P < 0.005). Conclusions: Small concentrations of ethanol (1 mM) induced apoptosis in HepG2 MCTS with a concomitant inhibition on outgrowth potential, accompanied with a low degree of necrosis. These findings suggest that low concentrations of ethanol may already be sufficient for the treatment of hepatocellular carcinoma. Received: 20 July 1999 / Accepted: 27 November 1999     

Study of the effect of vitamins C and E on the radiation response of multicell spheroids treated with adriamycin

Treatment with Adriamycin (0.6 kg/ml for 60 min) was not cytotoxic to multicell spheroids. At this concentration, the drug was not a sensitizer of hypoxic cells in V79 multicell spheroids, which were irradiated at 37°C in medium equilibrated with a mixture of 5% 02:3% C02:92% N₂. The addition of vitamins C and E did not increase the radiation sensitivity of Adriamycin-treated spheroids. In some experiments, catalase was included in the growth medium to overcome the toxic effect of hydrogen peroxide, which is known to be formed in solutions containing vitamin C and also in Adriamycin solutions treated with vitamin C or microsomal preparations. As a result of these experiments, it was found that catalase increased theradiation killing in multicell spheroids.     

Effects of lucanthone on chinese hamster V-79 cells I. Interaction with radiation in monolayers and spheroids

Lucanthone has previously been shown to reduce the capacity of cells to accumulate and repair sublethal radiation damage, which suggests its potential as an adjuvant to clinical radiotherapy. Present results indicate that lucanthone was effective even for V-79 cells having a markedly enhanced shoulder on the radiation survival curve as a result of growth as multicell spheroids. Lucanthone also proved to be an effective cytotoxic agent in large spheroids. Unlike radiation toxicity, however, lucanthone toxicity was equal for cells from asynchronous monolayers or small spheroids. Thus, for equal levels of drug toxicity, the radiation-modifying effect of lucanthone was greater in the spheroid; the “interaction” was larger in the system which had a greater capacity for accumulation and repair of sublethal radiation damage. Direct extrapolation of this result to the clinic would suggest that lucanthone be used judiciously, and that it might be of value as an adjuvant to radiotherapy only in the unusual circumstance where the malignant tissue has a larger capacity for accumulation and repair of sublethal radiation damage than the surrounding normal tissue.     

Drug and radiation resistance in spheroids: cell contact and kinetics

Cells from multicellular spheroids are often more resistant than monolayers to drugs and radiation. While explanations for resistance can be based on differences in cell cycle distribution, inability of the drug to penetrate the spheroid, or the presence of hypoxic cells, these mechanisms do not adequately explain resistance to all agents. Small spheroids (containing about 25–50 cells) exposed to ionizing radiation, hyperthermia, photodynamic therapy, or topoisomerase II inhibitors, are more resistant to killing than monolayers; the close three-dimensional contact in spheroids has been implicated in this resistance. Proposed mechanisms for the contact effect include gap junctional reciprocity, cell shape mediated changes in (repair-related) gene expression, and alterations in chromatin packaging which influence DNA repair. The consequences of the contact effect are especially important for multifraction exposures. Another form of resistance can be demonstrated during repetitive treatments; regrowth resistance reflects the capacity of spheroid cells to proliferate more efficiently to compensate for cell killing.     

Preclinical evaluation of a new liposomal formulation of cisplatin,lipoplatin, to treat cisplatin-resistant cervical cancer

Objective

Cisplatin-based chemotherapy has been shown to improve survival in cervical cancer; however, treatment is associated with tumor resistance and significant toxicity. Lipoplatin is a new liposomal formulation of cisplatin, developed to reduce cisplatin toxicity, to improve drug accumulation at tumor sites and to overcome drug resistance.The aim of this study is to analyze the antitumoral activity of lipoplatin against cisplatin-resistant cervical cancer cells and to investigate its mechanism of action.

Methods

The activity and mechanism of action of lipoplatin were studied in the ME-180 cervical cancer cell line and its cisplatin-resistant clone R-ME-180 and HeLa cells using cell proliferation assays, flow cytometry, ELISA assay, cell migration, spheroids and tumor xenograft.

Results

We demonstrated that lipoplatin exhibited a potent antitumoral activity on HeLa, ME-180 cells and its cisplatin-resistant clone R-ME-180. Lipoplatin inhibited cell proliferation in a dose-dependent manner and was more active than the reference drug cisplatin in R-ME-180 cells and induced apoptosis, as evaluated by Annexin-V staining and DNA fragmentation, caspases 9 and 3 activation, Bcl-2, and Bcl-xL down-regulation, but Bax up-regulation inhibited thioredoxin reductase (TrxR) enzymatic activity and increased reactive oxygen species (ROS) accumulation; reduced EGFR expression and inhibited both migration and invasion. R-ME-180, but not ME-180 cells, generated three-dimensional (3D)-multicellular spheroids expressing the cancer stem cell marker ALDH. The ability of R-ME-180 cells to form spheroids in vitro and tumors in nude mice was also remarkably decreased by lipoplatin.

Conclusions

Overall, our results suggest that lipoplatin has potential for the treatment of cisplatin-resistant cervical cancer.     

Cytochrome P450 activity in control and induced long-term cultures of rat hepatocyte spheroids

Long-term events such as enzyme induction or chronic toxicity require long-term liver culture models that maintain activity of xenobiotic metabolising enzymes. The levels of these enzyme activities and their responsiveness to chemical induction was studied in rat hepatocyte spheroids, a potential long-term hepatocyte culture model. In comparison with other long-term liver culture models, the basal metabolic activity of spheroids has not been well studied. Additionally, no existing data on the induction of CYP3A activity in spheroids could be found. The basal xenobiotic metabolising activity of rat hepatocyte spheroids was monitored over 14 days in culture, using testosterone as a probe substrate. When spheroids from days 2–14 in culture were compared to 24-h control spheroids, there was a differential maintenance of basal CYP activity. CYP2A and CYP3A activities were maintained over the culture period, while there were time-related decreases in CYP2C11 and CYP2C/CYP2B1/2 activities. The responsiveness of rat hepatocyte spheroids to chemical induction was studied following treatment with phenobarbitone (PB) or dexamethasone (DEX). PB treatment induced CYP2A, CYP2C, CYP2B1/2 and CYP3A activities. DEX treatment resulted in an induction of CYP3A and CYP2C11 activities. The results demonstrate that rat hepatocyte spheroids retained some of the liver-specific functions essential in a long-term hepatocyte culture model, thus making spheroids comparable to other long-term culture models available.