Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain.

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.

Increased vulnerability of neurones and glial cells to low concentrations of methylmercury in a prooxidant situation.

Using reaggregating rat brain cell cultures at two different stages of differentiation, we examined the biochemical effects of a 10-day treatment with nanomolar concentrations of methylmercuric chloride (monomethylmercury), in the presence or absence of promoters of hydroxyl radical formation (10 microM copper sulphate plus 100 microM ascorbate). A decrease in total protein content accounted for the general cytotoxicity of these compounds, whereas selective effects were assessed by determining the activities of cell type-specific enzymes. Methylmercury, up to 100 nM, as well as the copper ascorbate mixture, when applied separately, induced no general cytotoxicity, and only slight effects on neuronal parameters. However, when applying 100 nM methylmercury and the copper-ascorbate mixture together, a drastic decrease in neuronal and glial parameters was found. Under these conditions, the content of reactive oxygen species, assessed by 2',7'-dichlorofluorescin oxidation, increased greatly, while the activities of antioxidant enzymes decreased. In the presence of copper and ascorbate, differentiated cultures appeared more resistant than immature ones to low methylmercury concentrations (1-10 mM), but did undergo similar changes in both cell type-specific and antioxidant enzyme activities at 100 nM methylmercury. These results suggest that in prooxidant conditions low doses of mercury can become much more deleterious for the central nervous system.

Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

Biotechnological production of taxanes: a molecular approach

Plant cell cultures constitute a promise for the production of a high number of phytochemicals, although the majority ofbioprocesses that have been developed so far have not resultedcommercially successful. An overview indicates that most of theresearch carried out until now is of the empirical type. For this reason,there is a need for a rational approach to the molecular and cellularbasis of metabolic pathways and their regulation in order to stimulatefuture advances.The empirical investigations are based on the optimization of theculture system, exclusively considering input factors such as theselection of cellular lines, type and parameters of culture, bioreactordesign and elicitor addition, and output factors such as cellular growth,the uptake system of nutrients, production and yield. In a rationalapproach towards the elucidation of taxol and related taxaneproduction, our group has studied the relationship between the taxaneprofile and production and the expression of genes codifying forenzymes that participate in early, intermediate and late steps of theirbiosynthesis in elicited Taxus spp cell cultures. Our results show that elicitors induce a dramatic reprogramming of gene expression in Taxus cell cultures, whichlikely accounts for the enhanced production of taxol and related taxanes and we have alsodetermined some genes that control the main flux limiting steps. The application ofmetabolic engineering techniques for the production of taxol and taxanes of interest is also discussed.

Altered expression and posttranslational modification of neurofilaments in methylmalonic aciduria

Neurofilaments (NF), the main components of axonal cytoskeleton, are known to be involved in several neurodegenerative diseases. It has been reported that methylmalonate and propionate affect phosphorylation of NFs. In an in vitro model for methylmalonic aciduria our group has recently shown that 2- methylcitrate (2-MCA) is the most toxic metabolite for developing brain cells. Here, we studied the effects of repetitive administration of 1mM 2- MCA every 12 hours over 3 days on the development of NFs in 3D organotypic rat brain cell cultures. By immunohistochemistry with antibodies specific for the different NF subunits (light NFL, medium NFM, heavy NFH) as well as for phosphorylated (p) and glycosylated (g) forms of NFs, we observed a decrease of axonal labeling and a disorganized axonal pattern. Interestingly, signal retention of p-NFM and g-NFM was observed in neuronal soma. Western blotting showed the decrease of NFL and NFH subunits. Taken together, our data show that 2-MCA alters expression of the different NF subunits as well as their post-translational modifications. This likely results in disturbed NF assembly, abnormal accumulation of NF in neuronal cell bodies and impairment of axonal development.We conclude thatNF are involved in 2-MCA-induced neurodegeneration in methylmalonic aciduria.

Colorectal cancer metastasis: the DNA repair inhibitor Dbait increases sensitivity to hyperthermia and improves efficacy of radiofrequency ablation.

PURPOSE: To assess the usefulness of combining hyperthermia with a DNA repair inhibitor (double-strand break bait [Dbait]) and its potential application to radiofrequency ablation (RFA) in a preclinical model of human colorectal cancer. MATERIALS AND METHODS: The local ethics committee of animal experimentation approved all investigations. First, the relevance was assessed by studying the survival of four human colorectal adenocarcinoma cell cultures after 1 hour of hyperthermia at 41°C or 43°C with or without Dbait. Human colon adenocarcinoma cells (HT-29) were grafted subcutaneously into nude mice (n = 111). When tumors reached approximately 500 mm(3), mice were treated with Dbait alone (n = 20), sublethal RFA (n = 21), three different Dbait schemes and sublethal RFA (n = 52), or a sham treatment (n = 18). RFA was performed to ablate the tumor center alone. To elucidate antitumor mechanisms, 39 mice were sacrificed for blinded pathologic analysis, including assessment of DNA damage, cell proliferation, and tumor necrosis. Others were monitored for tumor growth and survival. Analyses of variance and log-rank tests were used to evaluate differences. RESULTS: When associated with mild hyperthermia, Dbait induced cytotoxicity in all tested colon cancer cell lines. Sublethal RFA or Dbait treatment alone moderately improved survival (median, 40 days vs 28 days for control; P = .0005) but combination treatment significantly improved survival (median, 84 days vs 40 days for RFA alone, P = .0004), with approximately half of the animals showing complete tumor responses. Pathologic studies showed that the Dbait and RFA combination strongly enhances DNA damage and coagulation areas in tumors. CONCLUSION: Combining Dbait with RFA sensitizes the tumor periphery to mild hyperthermia and increases RFA antitumor efficacy.

Differential expression of 240 kDa ConA-binding glycoprotein in vitro and in vivo detected by immunochemical methods.

The expression of the 240 ConA-binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained. The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cells cultures (mixed or neuron-enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.

Effect of the TAT-RasGAP(317-326) peptide on apoptosis of human malignant mesothelioma cells and fibroblasts exposed to meso-tetra-hydroxyphenyl-chlorin and light.

BACKGROUND: 5,10,15,20-Tetrakis(m-hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT) has shown insufficient tumor selectivity for the treatment of pleural mesothelioma. Tumor selectivity of mTHPC-PDT may be enhanced in the presence of the TAT-RasGAP(317-326) peptide which has the potential to specifically sensitize tumor cells to cytostatic agents. MATERIALS AND METHODS: H-meso-1 and human fibroblast cell cultures, respectively, were exposed to two different mTHPC doses followed by light delivery with and without TAT-RasGAP(317-326) administration. mTHPC was added to the cultures at a concentration of 0.04microg/ml and 0.10microg/ml, respectively, 24h before laser light illumination at 652nm (3J/cm(2), 40mW/cm(2)). TAT-RasGAP(317-326) was added to the cultures immediately after light delivery at a concentration of 20microM. The apoptosis rate was determined by scoring the cells displaying pycnotic nuclei. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Light delivery associated with 0.04microg/ml mTHPC resulted in a significantly higher apoptosis rate in the presence of TAT-RasGAP(317-326) than without in H-meso-1 cells (p<0.05) but not in fibroblasts. In contrast, 1.0microg/ml mTHPC and light resulted in a significantly higher apoptosis rate in both H-meso-1 cells and fibroblasts as compared to controls (p<0.05) but the addition of TAT-RasGAP(317-326) did not lead to a further significant increase of the apoptosis rate of both H-meso-1 cells and fibroblasts as compared to mTHPC and light delivery alone. CONCLUSION: TAT-RasGAP(317-326) selectively enhanced the effect of mTHPC and light delivery on H-meso-1 cells but not on fibroblasts. However, this effect was mTHPC dose-dependent and occurred only at a low sensitizer dose.

Characterization of the neuronal microtubule regulator SCG10 in transfected cells and " in vivo "

SUMMARY The ability of neuronal processes to find their way along complex paths and to establish appropriate connections depends on continual rearrangements of the cytoskeletal components. The regulation of microtubules plays an important role for morphological changes underlying nevrite outgrowth, axonal elongation, and growth cone steering. SCG10 (superior cervical ganglion clone 10) is a neuronal growthassociated protein developmentally regulated and highly enriched in the neuronal growth cones. SCG10 presents a microtubule destabilizing activity that could participate to the regulation of microtubule dynamics and thus explain microtubule behaviors in the growth cone during axonal elongation and turning. It is here suggested that a tight control of the opposite effects on microtubules of SCG10 and the stabilizing microtubule-associated protein MAP1B allows a fine tuning of cytoskeletal rearrangement and may provide the required microtubule dynamic instability to promote axonal growth. Moreover, antibodyblockade of SCG10 function, that leads to growth cone pauses similar as those triggered by the guidance molecule EphB, and the modulation of SCG10 activity by the Rho GTPase Rnd1 suggest a potential role for SCG10 in the signal transduction pathways of extracellular guidance cues. The identification of the active zone protein Bassoon as a potential interaction partner for the SCG10-related protein NPC2, using atomic force microscopy as well as COS-7 and neuronal cell cultures, also gives new insights for a role of this protein family into the processes of synapse genesis or plasticity. Finally, SCG10 mutant mice generated by gene targeting and expressing a soluble form of the protein have been characterized during early postnatal development and in the adulthood. Due to the deletion of its membrane binding domain, SCG10 specific subcellular targeting to growth cones is compromised and results in impairments of motor and coordination development. Further histological analysis in the sciatic nerve reveal that these symptoms are associated with neurodegenerative signs. RESUME Une navigation correcte des prolongements cellulaires neuronaux leur permettant de former des connections appropriées repose sur de continuels réarrangements des constituants de leur cytosquelette. La régulation des microtubules joue notamment un rôle important dans les changements morphologiques qui accompagnent la croissance axonale et les réorientations du cône de croissance. SCG10 (superior cervical ganglion clone 10) est une protéine étroitement associée à la croissance neuronale, hautement régulée durant le développement et abondante au niveau du cône de croissance. SCG10 présente une activité déstabilisatrice sur les microtubules qui pourrait permettre une régulation des paramètres dynamiques propres aux microtubules et ainsi expliquer leur comportement durant la navigation du cône de croissance. Il est ici proposé qu'un contrôle précis des effets opposés de SCG10 et d'une autre protéine stabilisante associée aux microtubules (MAP1 B) permette un réglage fin des réarrangements du cytosquelette et puisse ainsi produire l'instabilité dynamique nécessaire à la croissance anale. Par ailleurs, le blocage de la fonction de SCG10 par un anticorps spécifique, conduisant à des pauses du cônes de croissance similaires à celles provoquées par la molécule de guidage EphB, ainsi que la modulation de l'activité de SCG10 par la Rho GTPase Rnd1 suggèrent une potentielle implication de SCG10 dans les voies de transduction des signaux provenant de molécules de guidage extracellulaires. L'identification d'une interaction de la protéine synaptique Bassoon avec la protéine NPC2 apparentée à SCG10, au moyen de la microscopie à force atomique et dans des cultures de cellules neuronales et COS-7, ouvre des perspectives concernant ces protéines dans la formation et la plasticité synaptiques. Finalement, des souris mutantes pour SCG10 produites par ciblage de gène et exprimant une forme soluble de la protéine ont été caractérisées durant la phase précoce du développement et à l'âge adulte. La délétion du domaine permettant l'ancrage de SCG10 aux membranes compromet sa sub-localisation au niveau du cône de croissance et résulte en l'apparition de troubles moteurs et de la coordination. Des analyses histologiques complémentaires au niveau du nerf sciatique montrent que ces symptômes sont associés avec des signes neurodégénératifs.

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-β-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-β (Aβ) and the degeneration of photoreceptors. To explore the largely unknown role of Aβ in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aβ(1-42). Subretinal injection of the Aβ peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aβ did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aβ-induced photoreceptor apoptosis. Subretinal injection of Aβ was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aβ in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aβ-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aβ-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aβ-injected retinas. Our study pinpoints the roles of Aβ and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

Cancer chronotherapeutics: experimental, theoretical, and clinical aspects.

The circadian timing system controls cell cycle, apoptosis, drug bioactivation, and transport and detoxification mechanisms in healthy tissues. As a consequence, the tolerability of cancer chemotherapy varies up to several folds as a function of circadian timing of drug administration in experimental models. Best antitumor efficacy of single-agent or combination chemotherapy usually corresponds to the delivery of anticancer drugs near their respective times of best tolerability. Mathematical models reveal that such coincidence between chronotolerance and chronoefficacy is best explained by differences in the circadian and cell cycle dynamics of host and cancer cells, especially with regard circadian entrainment and cell cycle variability. In the clinic, a large improvement in tolerability was shown in international randomized trials where cancer patients received the same sinusoidal chronotherapy schedule over 24h as compared to constant-rate infusion or wrongly timed chronotherapy. However, sex, genetic background, and lifestyle were found to influence optimal chronotherapy scheduling. These findings support systems biology approaches to cancer chronotherapeutics. They involve the systematic experimental mapping and modeling of chronopharmacology pathways in synchronized cell cultures and their adjustment to mouse models of both sexes and distinct genetic background, as recently shown for irinotecan. Model-based personalized circadian drug delivery aims at jointly improving tolerability and efficacy of anticancer drugs based on the circadian timing system of individual patients, using dedicated circadian biomarker and drug delivery technologies.

BRAIN DAMAGE IN METHYLMALONIC ACIDURIA: 2-METHYLCITRATE LEADS TO AMMONIA INCREASE AND APOPTOSIS

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in methylmalonic aciduria. 1 mM methylmalonate (MMA), 2-methylcitrate (2-MCA) or propionate (PA) were repeatedly added to the culture media at two different time points of the cultures. In cultures treated with 2-MCA, we observed a significant increase of lactate in the medium, consistent with a possible inhibition of Krebs cycle and respiratory chain, as described earlier in the literature. Interestingly, we further observed that 2-MCA induced an important increase in ammonia production with concomitant decrease of glutamine concentrations, which suggests an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease with deleterious effects on early stages of brain development. By immunohistochemistry we could show that 2-MCA substantially increased the number of apoptotic cells. On the cellular level, 2-MCA had a toxic effect (cell swelling and cell death) on glial cells, but not on neurons. Surprisingly, MMA seemed to have a growth stimulating effect on the cultures. We can conclude that 2-MCA was the most toxic metabolite in our model for methylmalonic aciduria inducing ammonia accumulation and massive apoptosis in brain cells.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

Triodothyronine enhancement of neuronal differentiation in aggregating fetal rat brain cells cultured in a chemically defined medium.

Triiodothyronine (30 nM) added to serum-free cultures of mechanically dissociated re-aggregating fetal (15-16 days gestation) rat brain cells greatly increased the enzymatic activity of choline acetyltransferase and acetylcholinesterase throughout the entire culture period (33 days), and markedly accelerated the developmental rise of glutamic acid decarboxylase specific activity. The enhancement of choline acetyltransferase and acetylcholinesterase specific activities in the presence of triiodothyronine was even more pronouned in cultures of telencephalic cells. If triiodothyronine treatment was restricted to the first 17 culture days, the level of choline acetyltransferase specific activity at day 33 was 84% of that in chronically treated cultures and 270% of that in cultures receiving triiodothyronine between days 17 and 33, indicating that relatively undifferentiated cells were more responsive to the hormone. Triiodothyronine had no apparent effect on the incorporation of [3H]thymidine at day 5 or on the total DNA content of cultures, suggesting that cellular differentiation, rather than proliferation was affected by the hormone. Our findings in vitro are in good agreement with many observations in vivo, suggesting that rotation-mediated aggregating cell cultures of fetal rat brain provide a useful model to study thyroid hormone action in the developing brain.

The protective role of transferrin in Müller glial cells after iron-induced toxicity.

PURPOSE: Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection. METHODS: We generated transgenic mice (Tg) carrying the complete human transferrin gene on a C57Bl/6J genetic background. We studied human (hTf) and mouse (mTf) transferrin localization in Tg and wild-type (WT) C57Bl/6J mice using immunochemistry with specific antibodies. Müller glial (MG) cells were cultured from explants and characterized using cellular retinaldehyde binding protein (CRALBP) and vimentin antibodies. They were further subcultured for study. We incubated cells with FeCl(3)-nitrilotriacetate to test for the iron-induced stress response; viability was determined by direct counting and measurement of lactate dehydrogenase (LDH) activity. Tf expression was determined by reverse transcriptase-quantitative PCR with human- or mouse-specific probes. hTf and mTf in the medium were assayed by ELISA or radioimmunoassay (RIA), respectively. RESULTS: mTf was mainly localized in retinal pigment epithelium and ganglion cell layers in retina sections of both mouse lines. hTf was abundant in MG cells. The distribution of mTf and hTf mRNA was consistent with these findings. mTf and hTf were secreted into the medium of MG cell primary cultures. Cells from Tg mice secreted hTf at a particularly high level. However, both WT and Tg cell cultures lose their ability to secrete Tf after a few passages. Tg MG cells secreting hTf were more resistant to iron-induced stress toxicity than those no longer secreted hTf. Similarly, exogenous human apo-Tf, but not human holo-Tf, conferred resistance to iron-induced stress on MG cells from WT mice. CONCLUSIONS: hTf localization in MG cells from Tg mice was reminiscent of that reported for aged human retina and age-related macular degeneration, both conditions associated with iron deposition. The role of hTf in protection against toxicity in Tg MG cells probably involves an adaptive mechanism developed in neural retina to control iron-induced stress.