Cyclosporin A is routinely used in transplant therapy following allogeneic or xenogeneic tissue transplantation to prevent rejection. This immunosuppressive drug is also neurotoxic; however, its mechanisms of action for neurotoxicity are poorly understood. Undifferentiated and cyclic adenosine 3′,5′-monophosphate (cAMP)–induced differentiated neuroblastoma (NB) cells were used as an experimental model to study the toxicity of cyclosporin A. Results showed that cyclosporin A promoted the outgrowth of neurites and inhibited the growth of undifferentiated NB cells. When cyclosporin A was added simultaneously with RO20–1724, an inhibitor of cyclic nucleotide phosphodiesterase, or with prostaglandin E₁, a stimulator of adenylate cyclase, it markedly enhanced the growth inhibitory and differentiation effects of these cAMP-stimulating agents. In addition, cyclosporin A added to cAMP-induced differentiated NB cells caused dose-dependent degeneration of these cells as evidenced by the vacuolization of cytoplasm and the fragmentation of nuclear and cytoplasmic materials; however, neurites remained intact. Cyclosporin A alone did not alter the intensity of cell immunostaining for ubiquitin or β-amyloid peptide (amino acids 1–14) (Aβ_1–14); however, it enhanced the intensity of staining for both ubiquitin and Aβ in cells that were treated with cAMP-stimulating agents. The intensity of staining of amyloid precursor protein (amino acids 44–63) (APP_44–63) did not change in any treated group, suggesting that the increase in Aβ staining is due to increased processing of APP to Aβ. We propose that one of the mechanisms of cyclosporin A–induced neurotoxicity involves increased levels of Aβ and ubiquitin.

The teratogenic potential of two antifungal triazoles (Triadimefon and Triadimenol) has been investigated in vitro by the rat postimplantation whole embryo culture method. Rat embryos 9.5 d old were cultured for 48 h in rat serum with Triadimefon (12.5–250 μM) or Triadimenol (6.25–125 μM) and then examined. Some embryos exposed to Triadimenol (6.25–125 μM) were cultured for 12 extra hours in control serum to improve their developmental degree and then immunostain cranial nerves and ganglia. The exposure to the highest doses of triazoles only moderately reduced some morphometrical developmental parameters. By contrast, 25–250 μM Triadimefon and 25–125 μM Triadimenol induced specific concentration-related teratogenic effects at the level of first and second branchial arches. After immunostaining, embryos exposed to 12.5–125 μM Triadimenol showed specific cranial nerve and ganglia abnormalities. The possible implication of neural crest cell alterations on triazole-related abnormalities is discussed.

Many studies are being conducted to define the role of growth factors in cutaneous physiology in order to add cytokines in a timely fashion for optimal tissue engineering of skin. This study is aimed at developing a multistep approach for the production of bioengineered skin substitutes, taking into account the effects of various growth factors according to the culture time. The use of a serum-supplemented medium throughout the whole culture period of skin substitutes was compared to the sequential use of specific additives at defined culture steps. Histological analysis revealed that serum was necessary for keratinocyte proliferation and migration on dermal substitutes during the first 2 d after their seeding. However, the serum-free medium presented some advantages when supplemented with different additives at specific culture steps. Interestingly, ascorbic acid added to the dermal substitutes before and after keratinocyte seeding maintained their cuboïdal morphology in the basal epidermal layer. In the absence of serum, collagen matrix degradation slowed down, and a better multilayered epidermal organization was obtained, notably with retinoic acid. Stratum corneum formation was also enhanced by fatty acids. Thus, sequential addition of exogenous factors to the medium used to produce skin substitutes can improve their structural features and functional properties in vitro.

The ascites of a 78-yr-old Japanese woman with cholangiocarcinoma was used for a primary culture. An established new cell line (designated TK from the Japanese description of cholangiocarcinoma; Tankan-gann) showed conspicuous tumor marker production. A high level of circulating serum tumor markers; carbohydrate antigen (CA) 19-9, 32,000 U/ml; CA50, 6900 U/ml; and carcinoembryonic antigen (CEA), 300 ng/ml (on an average from 10⁶ cells/ml for 3 d culture) were detected in the tissue culture supernatant. With an inoculum of 2 × 10⁷ TK cells, nude mice progressively developed tumors. The histological features of the tumors forming in nude mice showed well-differentiated adenocarcinoma. Within the tumor mass, large amounts of extra cellular fluid retained approximately 590,000 U/ml of CA19-9, 200,000 U/ml of CA50, and 2000 ng/ml of CEA. Alpha-feto–protein was undetectable in the TK culture supernatant. There are few cholangiocarcinoma cell lines producing stable human tumor markers. Newly established TK cells derived from cholangiocarcinoma showed a stable production of serum tumor markers in vivo and in vitro. The changes in the tumor marker secretion ratios were shown to be dependent upon type of tumor cells, i.e., whether they are in vitro or in vivo. These features make TK cells a valuable tool for studying tumor markers.

We constructed a recombinant adenovirus vector that contained the origin-defective SV40 early gene, coding temperature-sensitive T antigen. This vector transferred the SV40 early gene into human epidermal keratinocytes with high efficiency. T antigen conferred the ability of keratinocytes to grow with limited differentiation in the presence of serum and high calcium concentration at the permissive temperature (34° C), although normal keratinocytes were induced to differentiate and stop growing under the same conditions. The serum/Ca -resistant cells did not proliferate at the nonpermissive temperature (40° C), indicating that they depended on T antigen for their proliferation. The temperature-sensitive T antigen dissociated from the tumor suppressor gene products, p53, at 40° C. The serum/Ca -resistant cells still had the ability to proceed to terminal differentiation when injected into SCID mice as cultured keratinocytes. However, they did not form an apparent basal layer. This indicated that the tissue remodeling process in the serum/Ca -resistant keratinocytes was abnormal. All of these epidermoid cysts disappeared within 8 wk and no tumor developed for 6 mo. We consider that ΔE1/SVtsT is a useful tool to examine multistep carcinogenesis of human epithelial cells in vitro.

Sf21 and Sf9 cell lines established from the lepidoptera Spodoptera frugiperda do not display major induction of heat shock proteins when exposed to a temperature of 37° C. After some months of adaptation at 37° C we obtained two new cell lines, Sf21-HT and Sf9-HT, which have now been established for several years in our laboratory. The Sf9-HT line displays a slightly shorter doubling time at 37° C than the wild type at 28° C, but cell lethality gives rise to an earlier growth arrest. The composition of total lipid extract from heat-adapted cells reveals a higher sphingomyelin to phosphatidylcholine ratio and a higher percentage of saturated fatty acids, which are expected for the lower membrane fluidity, required for thermotolerance. The cell volume of Sf9-HT is doubled, and by flow cytometry we showed that the DNA content is twice that in the parental cell line. Karyotypic examination of metaphasic cells achieved under epifluorescence microscopy revealed a doubled chromosome number in Sf9-HT.

The purpose of this study is to identify optimal culture conditions to support the proliferation of human macrovascular endothelial cells. Two cell lines were employed: human saphenous vein endothelial cells (HSVEC) and human umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) were investigated in relation to cell proliferation. Additionally, a variety of extracellular matrix (ECM) substrate–coated culture dishes were also tested. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulted in a 2.3-fold increase in cell proliferation. Media containing 20% FBS increased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 μg/ml) and endothelial cell growth supplement (ECGS) (50 μg/ml) significantly improved cell proliferation. Epithelial growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when endothelial cells were grown on uncoated culture plates compared to plates coated with ECM proteins: fibronectin, laminin, gelatin, or collagen types I and IV. The culture environment yielding maximal HSVEC and HUVEC proliferation is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 50 μg/ml heparin, and 50 μg/ml ECGS. The ECM substrate–coated culture dishes offer no advantage.

Ouabain, a specific inhibitor of the sodium- and potassium-activated adenosine triphosphatase, causes reversible inhibition of the fusion of myoblasts to form myotubes. We further examined this observation to investigate whether control of Na/K-ATPase activity may normally contribute to the regulation of myogenesis. In control cultures, fusion was preceded by a small decrease in intracellular sodium concentration, but intracellular sodium and potassium increased significantly during fusion. Levels of ouabain that produce prolonged inhibition of fusion (400 μM) virtually eliminated sodium and potassium gradients. However, lower ouabain levels (10–100 μM) also produced significant changes in intracellular potassium and/or sodium along with little apparent decrease in the eventual extent of fusion. The effect of ouabain on protein synthesis was also examined. Low levels of ouabain (<50 μM) that did not affect myogenesis also did not affect incorporation of radiolabeled amino acids, while higher concentrations produced a decline in protein synthesis that paralleled decreases in the rate of myoblast fusion. Levels of metabolic labeling were reduced 90% in cultures treated with 400 μM ouabain. Inhibition of protein synthesis would prevent membrane remodeling required for fusion and other events in myogenesis. Thus, our results do not support any specific role for the sodium- and potassium-activated adenosine triphosphatase in regulating myogenesis.

Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 showed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation pattern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90α or -β isoform via the gene transfer method. On the other hand, the overexpression of HSP90β alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiologically critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.