Experimental confounding factors affecting stability, transport and metabolism of flavanols and hydroxycinnamic acids in Caco-2 cells.

Studying bioavailability of polyphenols is essential to understand the health effects of these compounds. Human epithelial cells are commonly used in intestinal absorption and transport experiments but the changes polyphenols undergo during incubation, due to their chemical instability under the cell culture conditions, are scarcely known and might lead to inaccurate conclusions. Based on abundance of flavanols and hydroxycinnamic acids in the diet, epicatechin, epicatechin-3-gallate and procyanidin B2 as flavanols along with 5-caffeoylquinic and 3,5-dicaffeoylquinic acids as hydroxycinnamic acids were selected to comparatively evaluate their absorption and metabolism using an in vitro Caco-2 cell model. Special emphasis was paid to the structure-stability relationship of these phenolic compounds in Dulbecco's Modified Eagle's Medium (DMEM) under the cell culture conditions. The tested compounds were scarcely absorbed and minimally metabolized by the intestinal epithelium cells. The cell transport study showed prevalent efflux for flavanols opposite to absorption for hydroxycinnamates. Intestinal metabolism revealed that hydroxycinnamates were preferentially hydrolyzed and subsequently methylated, whereas hydrolysis of flavanols could not be confirmed, being mostly conjugated to sulfate, methyl- and methyl-sulfate derivatives. It is noteworthy that methyl derivatives of procyanidin-B2 were detected inside Caco-2 cells, confirming its absorption. In addition, culture medium influenced phenol isomerization to a higher extent than cells. In conclusion, hydroxycinnamates were better absorbed than flavanols although their bioavailability was limited in this intestinal cell model.

Glucagon-like peptide-1 improves beta-cell antioxidant capacity via extracellular regulated kinases pathway and Nrf2 translocation.

Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation.

Protein tyrosine phosphatase 1B modulates GSK3ß/Nrf2 and IGFIR signaling pathways in acetaminophen-induced hepatotoxicity.

Acute hepatic failure secondary to acetaminophen (APAP) poisoning is associated with high mortality. Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of tyrosine kinase growth factor signaling. In the liver, this pathway confers protection against injury. However, the involvement of PTP1B in the intracellular networks activated by APAP is unknown. We have assessed PTP1B expression in APAP-induced liver failure in humans and its role in the molecular mechanisms that regulate the balance between cell death and survival in human and mouse hepatocytes, as well as in a mouse model of APAP-induced hepatotoxicity. PTP1B expression was increased in human liver tissue removed during liver transplant from patients for APAP overdose. PTP1B was upregulated by APAP in primary human and mouse hepatocytes together with the activation of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), resulting in cell death. Conversely, Akt phosphorylation and the antiapoptotic Bcl2 family members BclxL and Mcl1 were decreased. PTP1B deficiency in mouse protects hepatocytes against APAP-induced cell death, preventing glutathione depletion, reactive oxygen species (ROS) generation and activation of JNK and p38 MAPK. APAP-treated PTP1B(-/-) hepatocytes showed enhanced antioxidant defense through the glycogen synthase kinase 3 (GSK3)ß/Src kinase family (SKF) axis, delaying tyrosine phosphorylation of the transcription factor nuclear factor-erythroid 2-related factor (Nrf2) and its nuclear exclusion, ubiquitination and degradation. Insulin-like growth factor-I receptor-mediated signaling decreased in APAP-treated wild-type hepatocytes, but was maintained in PTP1B(-/-) cells or in wild-type hepatocytes with reduced PTP1B levels by RNA interference. Likewise, both signaling cascades were modulated in mice, resulting in less severe APAP hepatotoxicity in PTP1B(-/-) mice. Our results demonstrated that PTP1B is a central player of the mechanisms triggered by APAP in hepatotoxicity, suggesting a novel therapeutic target against APAP-induced liver failure.

[Characterization of cocoa fiber and its effect on the antioxidant capacity of serum in rats]. / Caracterización de la fibra de cacao y su efecto sobre la capacidad antioxidante en suero de animales de experimentación.

OBJECTIVES: The aim of this study was to characterize the physico-chemical properties of cocoa fibre (CF), to analyze its polyphenolic content and antioxidant capacity in vitro, and to investigate the effect of the administration of a polyphenolic extract of this cocoa fiber on the antioxidant capacity of the serum in rats. METHODS AND MATERIALS: Dietary fiber (DF) composition and polyphenolic (PP) content of the cocoa fiber were analyzed. The antioxidant capacity of the CF was determined by means of its reduction power (FRAP) and the capacity to scavenge free radicals (ABTS). To evaluate the bioavailability and the antioxidant capacity in vivo of the phenolic compounds of CF, an extract of these compounds was administred in the stomach of the rats with a gastric probe (100 mg PP/kg), taking blood samples at different time intervals. Sera were analyzed by HPLC to determine the presence/absence of PP or PP-metabolites. In orther to evaluate the antioxidant capacity of the serum FRAP and ABTS methods were used. RESULTS: Cocoa fiber was an excellent source of DF, with a high content of total dietary fiber (TDF), over 60% of the dry matter, made up mainly of insoluble dietary fiber (IDF; 83% of TDF). This fiber had just 1.15% of polyphenols, with low antioxidant activity. After intragastric administration of the PP-rich fraction a fast and measurable absorption of the CF polyphenols was observed, being epicatechin the main PP in blood. The absorption of this PP confers a significant, although transitory increase of the serum antioxidant capacity 10-45 minutes post-gavage; after this time, the antioxidant capacity progressively decreased reaching basal levels after 6 h. CONCLUSIONS: Cocoa fiber can be considered as an excellent source of DF, mainly insoluble dietary fiber; therefore, it could be used as an ingredient in fiber-rich functional foods. Besides the benefits derived from its high fiber content, the CF would provide protection against oxidative damage by means of its content in phenolic compounds (epicatechin) wich are absorbed maintaining the antioxidant properties in vivo.

Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation.

Aplidin is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin-induced JNK activation and cytotoxicity. Our results show that Aplidin induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.

Steroid and protein regulators of glial cell proliferation.

Any variation in CNP and GS activity in vitro under the effect of growth factors with different mechanisms of action, may indicate a general process of differentiation in the culture. An increase in the GS activity in early passage glioma may be interpreted as an induction of the differentiating phenotype involving cell transformation into a more astrocytic-like culture. Hence, under the above experimental conditions, dexamethasone and insulin are the two factors with the most potent differentiating effect on rat C6 glioma cell line. Comparative studies with growth regulators using different receptor pathways and mechanisms of action may add valuable data to the current knowledge in the field. The data presented in this chapter show the plasticity of C6 glioma to the differentiating effect of various growth factors, especially insulin and dexamethasone, and confirms C6 glioma cell line as a useful model for studies on glial cell properties and proliferation in vitro.

Effect of growth factors on the in vitro growth and differentiation of early and late passage C6 glioma cells.

The effect of different hormones and growth factors was assayed on the in vitro growth and enzymatic activities of 2',3'-cyclic nucleotide 3'phosphohydrolase (CNP) and glutamine synthetase (GS) of rat glioma C6 cells at two different passages in culture. Young cultures (passage 26), mainly oligodendrocytic, and older cultures (passage 134), predominantly astrocytic, were treated with 10 microM dexamethasone, 20 ng/ml transforming growth factor alpha (TGF alpha), 10 ng/ml insulin, 20 ng/ml platelet-derived growth factor (PDGF), and 20 ng/ml, epidermal growth factor (EGF) in serum-free chemically defined media. In vitro growth rate was measured in terms of DNA content, by a fluorometric method of diaminobenzoic acid, and rate of DNA synthesis by 3H-thymidine incorporation. CNP activity (marker for in vitro oligodendrocytes) and GS activity (marker for astrocytes) were determined spectrophotometrically. Dexamethasone reversibly and significantly inhibited growth of C6 glioma in early and late passages. PDGF and insulin promoted in vitro growth only in late passage but not in early passage cells, whereas EGF and TGF alpha did not significantly affect growth. An increase in CNP activity was observed in early passage cells under the effect of PDGF and insulin. The increase in GS activity induced by insulin and dexamethasone suggests a differentiating role for these factors in C6 glioma cells. These results further present the C6 glioma cell line as a useful model for studies on glial cell properties and responsiveness in culture and support its use in experimental aging in vitro.

Glucocorticoids induce a G1/G0 cell cycle arrest of Con8 rat mammary tumor cells that is synchronously reversed by steroid withdrawal or addition of transforming growth factor-alpha.

Con8 mammary tumor cells are an epithelial cell line derived from the 7,12-dimethylbenz(alpha)anthracene-induced 13762NF rat mammary adenocarcinoma. The synthetic glucocorticoid dexamethasone suppresses the growth of Con8 cells, and after 5 days of treatment with this steroid, Con8 cells undergo less than 0.5 population doublings. This growth arrest is accompanied by a 30-fold elevation in c-jun transcript levels, no change in c-fos expression, and a moderate increase in total AP-1 transcriptional activity. Dexamethasone inhibited DNA synthesis within one cell cycle, and flow cytometry of propidium iodide-stained nuclei demonstrated that dexamethasone growth-suppressed cells had a DNA content indicative of a specific cell cycle block in either G1 or G0. Consistent with a G1/G0 arrest of the cell cycle, dexamethasone did not prevent Con8 cells from entering the S phase after release from synchronization at the G1/S boundary by a double thymidine block. Analysis of [3H]thymidine incorporation and autoradiography of [3H]thymidine-labeled nuclei revealed that after either dexamethasone withdrawal or the addition of transforming growth factor-alpha (TGF alpha), Con8 cells synchronously reinitiate cell cycle progression. Northern blot analysis demonstrated that an induction of transcripts for the G1 marker genes c-myc and cyclin D1 occurs before cells enter the S-phase. After dexamethasone withdrawal, c-myc and cyclin D1 expression transiently peak at 2 and 4 h, respectively. In contrast, c-myc expression peaked at 0.5-1 h, whereas cyclin D1 expression was induced at 2 h and maintained at a high level after the addition of TGF alpha. Our results demonstrate that glucocorticoids induce a specific block of the cell cycle progression of a rat mammary tumor cell, and that after synchronous progression through the cell cycle, the temporal expression pattern for c-myc and cyclin D1 is distinct for dexamethasone release vs. the addition of TGF alpha to glucocorticoid-suppressed cells.

Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase.

Serine/threonine protein kinases are important regulators of diverse cellular processes including metabolism, proliferation, and differentiation. We have previously identified the cDNA for a 49-kDa serine/threonine kinase, designated sgk, which is transcriptionally responsive to glucocorticoid hormones and serum in epithelial cells. We report here that sgk expression is also rapidly induced by dexamethasone or serum in Rat2 fibroblasts. Nuclear run-on and Northern blot analysis revealed that the induction of sgk mRNA is an immediate-early transcriptional response to serum stimulation of quiescent fibroblasts, which occurs just after the peak in c-jun transcription. In contrast to the glucocorticoid-stimulated sgk expression in Rat2 fibroblasts, the transcriptional induction of sgk by serum was transient and sgk transcripts decayed with a particularly rapid half-life of 20 min. The rapid turnover of sgk, in combination with its immediate-early transcriptional response to serum, suggests a novel mechanism for responding to mitogenic signals during G0 to S transition and entry into the cell cycle.

Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum.

A novel member of the serine/threonine protein kinase gene family, designated sgk, for serum and glucocorticoid-regulated kinase, was identified in a differential screen for glucocorticoid-inducible transcripts expressed in the Con8.hd6 rat mammary tumor cell line. sgk encodes a protein of 49 kDa which has significant sequence homology (45 to 55% identity) throughout its catalytic domain with rac protein kinase, the protein kinase C family, ribosomal protein S6 kinase, and cyclic AMP-dependent protein kinase. sgk mRNA is expressed in most adult rat tissues, with the highest levels in the thymus, ovary, and lung, as well as in several rodent and human cell lines. sgk mRNA was stimulated by glucocorticoids and by serum within 30 min, and both inductions were independent of de novo protein synthesis. The transcriptional regulation by glucocorticoids is a primary response, since the promoter of sgk contains a glucocorticoid response element consensus sequence 1.0 kb upstream of the start of transcription which is able to stimulate chloramphenicol acetyltransferase reporter gene activity in a dexamethasone-dependent manner. Antibodies that specifically recognize sgk-encoded protein on an immunoblot were generated. This protein was shown to increase in abundance with glucocorticoid treatment in a manner which paralleled the mRNA accumulation. This is the first report of a presumed serine/threonine protein kinase that is highly regulated at the transcriptional level by glucocorticoid hormones and suggests a novel interplay between glucocorticoid receptor signalling and a protein kinase of the second messenger family.

Glucocorticoids coordinately disrupt a transforming growth factor alpha autocrine loop and suppress the growth of 13762NF-derived Con8 rat mammary adenocarcinoma cells.

We have demonstrated previously that the synthetic glucocorticoid dexamethasone suppresses the growth of Con8 rat mammary tumor cells, which are derived from the 13762NF transplantable, hormone-responsive rat mammary adenocarcinoma. Dexamethasone inhibited [3H]thymidine incorporation into Con8 cells at high cell density under both serum and serum-free conditions. Fractionation in nonreducing sodium dodecyl sulfate-polyacrylamide gels of proteins secreted from dexamethasone-treated and untreated Con8 mammary tumor cells revealed two size classes of glucocorticoid inhibited mitogenic activities; a larger M(r) 27,000-33,000 and a smaller M(r) 5,000-12,000 activity. Both size classes of mitogens restimulated the growth of glucocorticoid-suppressed Con8 cells suggesting that they can act in an autocrine fashion. The smaller mitogen was identified as transforming growth factor alpha (TGF-alpha) since this activity competed with 125I-epidermal growth factor (EGF) for EGF receptor binding and was selectively immunodepleted with monoclonal TGF-alpha antibodies but not with EGF antibodies. Western blots and radioreceptor assay of Con8-secreted proteins revealed that glucocorticoids inhibited the production of a M(r) 5500 immunoreactive TGF-alpha protein by 10-fold. Consistent with a steroid effect on the level of TGF-alpha production, rather than on its activity, the specific mitogenic activities of the TGF-alpha s secreted by dexamethasone-treated and untreated Con8 cells were identical to that of recombinant human TGF-alpha. Treatment of intact cells with suramin, which dissociates ligand-receptor complexes, revealed that the EGF receptor-mediated mitogenic response is functional in both glucocorticoid-treated and untreated cells. Taken together, our results demonstrate that glucocorticoids suppress Con8 mammary tumor cell growth and disrupt a potential TGF-alpha autocrine loop which results in a dramatic reduction in the level of extracellular TGF-alpha.

Overexpression of transforming growth factor alpha overrides the glucocorticoid-mediated suppression of Con8 mammary tumor cell growth in vitro and in vivo.

In a preceding paper (D. B. Alexander et al., Cancer Res., 53: 1808-1815, 1993), we demonstrated that the in vitro glucocorticoid inhibition of Con8 mammary tumor cell growth is accompanied by the disruption of a transforming growth factor alpha (TGF-alpha) autocrine loop. This growth suppression response functions in vivo since proliferation of Con8-derived tumors was inhibited in rats treated with the synthetic glucocorticoid, dexamethasone. The effect of dexamethasone on Con8-derived tumor growth was reversible in that tumors rapidly grew at the site of inoculation after discontinuing injections of dexamethasone. To test the in vivo relationship between the glucocorticoid growth suppression response and the TGF-alpha autocrine loop, Con8 cells were transfected with a TGF-alpha expression vector and single cell-derived neomycin-resistant subclones were recovered. [3H]Thymidine incorporation of cultured monolayers of transfected Con8 mammary cells and measurement of tumor diameters in rats revealed that dexamethasone failed to suppress the in vitro proliferation or in vivo tumor growth of Con8-derived cells producing high constitutive levels of secreted TGF-alpha. In contrast, both the in vivo and in vitro growth of Con8 cells transfected with vector controls were fully suppressible by glucocorticoids. Consistent with our in vitro observations, these results demonstrate that the regulation of TGF-alpha production plays a key role in the in vivo glucocorticoid suppression of Con8-derived mammary tumor growth.

Glucocorticoids reversibly arrest rat hepatoma cell growth by inducing an early G1 block in cell cycle progression.

We have previously documented that glucocorticoids suppress the proliferation of BDS1 hepatoma cells, a rat epithelial tumor cell line derived from minimal deviation Reuber H35 hepatoma cells. Flow cytometry demonstrated that, after treatment with the synthetic glucocorticoid dexamethasone, the growth of an asynchronous population of BDS1 cells was arrested within one cell cycle which resulted in an accumulation of cells with a G1-G0-like DNA content. Consistent with a glucocorticoid-induced block early in the G1 phase of the cell cycle, propidium iodide flow cytometry revealed that addition of dexamethasone up to 2 h after release from contact inhibition prevented BDS1 hepatoma cells from entering S phase, whereas dexamethasone treatment after 2 h had no effect on the entry of cells into S phase. Moreover, dexamethasone treatment did not prevent BDS1 cells from entering S phase after release from synchronization at the G1-S boundary by a double thymidine block. Analysis of DNA content, [3H]-thymidine incorporation, and autoradiography of [3H]-thymidine-labeled nuclei revealed that, after release from dexamethasone, BDS1 cells synchronously reinitiated cell cycle progression and entered S phase 8 h after hormone withdrawal. Northern blot analysis demonstrated that the level of transcripts encoding the G1 marker genes CYL-1 and CYL-2 G1 cyclins peaked 4 h after dexamethasone withdrawal. Dexamethasone induced a 20-fold increase in the level of c-jun mRNA which was reversed after hormone withdrawal, whereas expression of c-fos transcripts remained at a low level during the time course of hormone treatment and withdrawal. Transient transfections with a collagenase-chloramphenicol acetyltransferase reporter gene showed that dexamethasone inhibited 12-O-tetradecanoylphorbol-13-acetate-inducible, but not basal, AP-1 transcription factor activity. Our results demonstrate that glucocorticoids reversibly induce an early G1 block in cell cycle progression of an epithelial tumor cell line that occurs with a coordinate elevation in the expression of c-jun transcripts.

Alterations in the growth and protein content of human neuroblastoma cells in vitro induced by thyroid hormones, stress and ageing.

The effects of the thyroid hormone triiodothyronine (T3), nerve growth factor (NGF) and stress (exposure to heat or aluminum sulfate) on growth, development and ageing of human neuroblastoma cells were studied in vitro. Differentiation of cells using retinoic acid and NGF inhibits cell growth and proliferation; simultaneously, it promotes acquisition of neuronal phenotype, down-regulation of T3 receptors, and an increase in catecholaminergic tyrosine hydroxylase activity and microtubule assembly. The actions of T3 on neuronal differentiation resemble those of NGF and suggest the existence of NGF-T3 interactions. Exposure to stress inhibits cell growth and proliferation, increases immunoreactivity to the microtubule-assembling protein tau (which occurs in paired filaments of neurofibrillary tangles in the aged human brain), and facilitates formation of tau-ubiquitin complexes (which also occur in the aged brain). Stress does not prevent the inhibition of cell proliferation by high doses of T3; however, T3 doses that are equivalent to physiological levels reduce stress-induced inhibition of growth. Previous studies have shown that stress may also induce in these cells facsimile lesions of normal and abnormal ageing, such as accumulation of lipofuscin pigments, formation of paired helical filaments and increased immunoreactivity to tau, beta-amyloid proteins, and ubiquitin. These lesions may represent cellular and molecular manifestations of increased vulnerability and susceptibility to genetic and extrinsic factors (e.g. hormones and environmental influences) with ageing. It is proposed that neuroblastoma cells may serve as a model to study mechanisms of neuronal ageing and to identify agents and conditions capable of preventing, delaying or reducing metabolic abnormalities leading to age-associated disorders.

In vivo effects of dexamethasone on the tumor growth of glucocorticoid-sensitive Fu5-derived rat hepatoma cells.

We have previously demonstrated that BDS.1 cells are a minimal deviation rat hepatoma cell line that is hypersensitive to the anti-proliferative effects of glucocorticoids in vitro. When transplanted into athymic mice, exposure to dexamethasone reduced the initial growth rate and increased the latency time before detection of palpable BDS.1-derived tumors but did not affect the maximal growth rate, size and histology of the tumor. After collagenase dissociation, the in vitro growth of BDS.1-derived tumor cells was fully suppressed by dexamethasone. Exposure to insulin prevented the glucocorticoid inhibition of anchorage-independent growth of BDS.1 cell colonies in vitro and may therefore be one of the systemic factors that masks the long term in vivo growth response of glucocorticoids.

Characterization of nuclear T3 receptors in human neuroblastoma cells SH-SY5Y: effect of differentiation with sodium butyrate and nerve growth factor.

Nuclear receptors for the thyroid hormone triiodothyronine (T3) have been identified in vivo in brain tissues and in vitro in mouse and rat neuroblastoma and glioma cells. The present study characterizes nuclear T3 receptors in human neuroblastoma SH-SY5Y cells and compares their level before and after differentiation. Undifferentiated cells, grown in DME/HAM F-12 medium supplemented with 10% fetal calf serum, show an abundant single type of nuclear receptor, indicated by a straight Scatchard plot, with a Kd of 0.11 nmol/l. After treatment with sodium butyrate (0.5 mM for 4 days) or NGF (2 nM for 6 days), the cells showed neuronal-like patterns (extension of neurites, slowing of growth, increased tyrosine hydroxylase activity), with a decrease in the number of nuclear T3 receptors. As sodium butyrate and NGF treatments differentiate neuroblastoma SH-SY5Y cells, these data suggest a down-regulation of T3 receptors with cell maturation.

Effects of serotonin on tyrosine hydroxylase and tau protein in a human neuroblastoma cell line.

The direct effects of the neurotransmitter serotonin on the catecholaminergic enzyme, tyrosine hydroxylase and the microtubule-associated tau protein were studied in a human neuroblastoma cell line. Undifferentiated LAN-5 cells, cultured in serum supplemented basal medium, or cells induced to differentiate by 6 day exposure to 10 uM retinoic acid were treated for 48 hr with 50 nM and 50 uM serotonin. In undifferentiated cells, serotonin treatment (50 uM) decreased both tyrosine hydroxylase activity and a 50 kD cytoplasmic fraction tau protein while 50 nM serotonin treatment caused this 50 kD protein to increase in the cytoplasmic fraction but decrease in the membrane fraction. While basal tyrosine hydroxylase activity increased in differentiated vs. undifferentiated cells, serotonin treatment had no effect on the enzyme or tau in differentiated LAN-5. This study shows serotonin to have direct effects on the biochemistry and cytoskeleton of undifferentiated cultured human neuroblastoma.