Low-serum Medium Research Articles

Impaired nuclear functions lead to increased senescence and inefficient differentiation in human myoblasts with a dominant p.R545C mutation in the LMNA gene

We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.

The influence of RGD-bearing hydrogels on the re-expression of contractile vascular smooth muscle cell phenotype

This study reports on the ability of poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with pendant integrin-binding GRGDSP peptides (RGD-gels) to support the re-differentiation of cultured vascular smooth muscle cells (SMCs) toward a contractile phenotype. Human coronary artery SMCs were seeded on RGD-gels, hydrogels with other extracellular matrix derived peptides, fibronectin (FN) and laminin (LN). Differentiation was induced on RGD-gels with low serum medium containing soluble heparin, and the differentiation status was monitored by mRNA expression, protein expression, and intracellular protein organization of the contractile smooth muscle markers, smooth muscle α-actin, calponin, and SM-22α. RGD-gels supported a rapid induction (2.7- to 25-fold up-regulation) of SMC marker gene mRNA, with expression levels that were equivalent to FN and LN controls. Marker protein levels mirrored the changes in mRNA expression, with levels on RGD-gels indistinguishable from FN and LN controls. Furthermore, these markers co-localized in stress fibers within SMCs on RGD-gels suggesting the recapitulation of a contractile apparatus within the cells. These results indicate that SMCs cultured on RGD-bearing hydrogels can re-differentiate toward a contractile phenotype suggesting this material is an excellent candidate for further development as a bioactive scaffold that regulates SMC phenotype.

Endothelial cells incubated with platelet‐rich plasma express PDGF‐B and ICAM‐1 and induce bone marrow stromal cell migration

Platelet-rich plasma (PRP) is used to accelerate bone repair through the growth factors released by platelets. The purpose of this study was to evaluate if PRP induce human umbilical vein endothelial cells (HUVEC) to express mRNA for osteogenic growth factors and stimulate the migration of bone marrow stromal cell (BMSC). The effects of PRP were compared to those induced by vascular endothelial growth factor-A (VEGF-A) or, as a negative control, by platelet poor plasma (PPP). After incubation with PRP, but not with PPP, HUVEC showed an increased expression of mRNA for platelet derived growth factor-B (PDGF-B), and this effect was not inhibited by an anti-VEGF-A antibody. The migration of BMSC was more stimulated by HUVEC incubated with PRP than by HUVEC incubated with low serum medium or PPP. Besides, PRP increased the expression of intercellular adhesion molecule-1 (ICAM-1) and osteoprotegerin, but did not affect the expression either of the receptor activator for nuclear factor kappaB ligand (RANKL) or of RANK. These findings support the hypothesis that PRP contribute to bone repair by favoring the pro-osteogenic function of endothelial cells, including the recruitment of osteoblast precursors and the expression of adhesion molecules for monocyte/macrophages, while inhibiting their pro-osteolytic properties.

Regulation of heme oxygenase-1 expression and MAPK pathways in response to kaempferol and rhamnocitrin in PC12 cells

Oxidative stress has been considered as a major cause of cellular injuries in a variety of clinical abnormalities, especially neural diseases. Our aim of research is to investigate the protective effects and mechanisms of kaempferol and rhamnocitrin (kaempferol-7-methyl ether) on oxidative damage in rat pheochromocytoma PC12 cells induced by a limited supply of serum and hydrogen peroxide (H2O2). The current result demonstrated that kaempferol protected PC12 cells from serum deprivation-induced apoptosis. Pretreatment of cells with kaempferol also diminished intracellular generation of reactive oxygen species (ROS) in response to H2O2 and strongly elevated cell viability. RT-Q-PCR and Western blotting revealed that kaempferol and rhamnocitrin significantly induced heme oxygenase (HO)-1 gene expression. Addition of zinc protoporphyrin (Znpp), a HO-1 competitive inhibitor, significantly attenuated their protective effects in H2O2-treated cells, indicating the vital role of HO-1 in cell resistance to oxidative injury. While investigating the signaling pathways responsible for HO-1 induction, we observed that kaempferol induced sustained extracellular signal-regulated protein kinase 1/2 (ERK1/2) in PC12 cells grown in low serum medium; while rhamnocitrin only stimulated transient ERK cascade. Addition of U0126, a highly selective inhibitor of MEK1/2, which is upstream of ERK1/2, had no effect on kaempferol- or rhamnocitrin-induced HO-1 mRNA expression, indicating no direct cross-talk between these two pathways. Furthermore, both kaempferol and rhamnocitrin were able to persistently attenuate p38 phosphorylation. Taking together, the above findings suggest that kaempferol and rhamnocitrin can augment cellular antioxidant defense capacity, at least in part, through regulation of HO-1 expression and MAPK signal transduction.

Estrogen receptor α is a target of mTOR/S6K1 signaling in control of breast cancer cell proliferation.

Abstract Abstract #4062 Background: The 40S ribosomal S6 kinase 1 (S6K1) acts downstream of the mammalian Target of Rapamycin (mTOR), and is sensitive to inhibition by rapamycin. Chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, and S6K1 is overexpressed in about 30% of cases. Overexpression of S6K1 correlates with increased rapamycin sensitivity. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the reason for high-level amplification of the RPS6KB1 gene specifically in breast cancer, and not other cancer types, and the identity of breast cell-specific S6K1 targets is not well understood. In this study, we report that S6K1 regulates growth of breast cancer cells by phosphorylating Estrogen Receptor α (ERα), and co-expression of S6K1 and ERα in breast cancer cells renders them sensitive to combination therapy with rapamycin and tamoxifen.
 Materials and Methods: Using immunoblot analysis we determined expression levels of S6K1 in breast cancer cell lines, and the phosphorylation status of ERα on Ser167 after serum or mitogen stimulation. We compared the effect of low-serum media, and rapamycin and/or 4-hydroxytamoxifen (4-HT) addition on breast cancer cell proliferation using neutral red uptake assay.
 Results: We determined that overexpression of S6K1 in breast cancer cells confers proliferative advantage in low serum, contributing to neoplastic transformation. Next, we wished to identify targets of S6K1 in control of proliferation of breast cancer cells. One of the proteins we identified as a target of S6K1 is ERα. ERα is frequently co-overexpressed with S6K1 in breast cancer cells, which could be an indication of co-selection, and ERα contains a putative S6K1 phosphorylation motif RERLAS167. We confirmed that ERα phosphorylation may be mediated by S6K1. We shown that in S6K1-overexpressing MCF7 cells, serum-stimulated ERα phosphorylation is rapamycin-sensitive. Moreover, ERα phosphorylation stimulated by insulin and tumor-promoting phorbol esters is also rapamycin-sensitive. S6K1 can phosphorylate ERα in an in vitro kinase assay. Lastly, shRNA-mediated suppression of S6K1 expression in MCF7 cells reduces ERα phosphorylation. Subsequently, we examined whether co-overexpression of S6K1 and ERα in breast cancer cells sensitizes them to combination therapy with rapamycin and tamoxifen. We analyzed proliferation of cells treated with rapamycin and/or 4-HT. We observed that in S6K1- and ERα-overexpressing cells, treatment with rapamycin and 4-HT resulted in greater inhibition of cell growth.
 Discussion: Our data suggest that one of the targets of S6K1 signaling in breast cancer cells is ERα, and together, these proteins promote breast cancer cell proliferation. The ultimate objective would be to determine whether combined inhibition of S6K1 and the estrogen receptor could be used as targeted therapy for patients with double positive expression of S6K1 and ER. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4062.

Efficient expansion of mesenchymal stromal cells from umbilical cord under low serum conditions

Mesenchymal stromal cells (MSC) are of clinical interest for their potential use in regenerative medicine and immunotherapy. Originally derived from bone marrow (BM), MSC have now been isolated from most tissues, including umbilical cord (UC) and UC blood (UCB). If MSC from UC are biologically equivalent to those from BM, they would be attractive as a readily available and non-invasive source for cellular therapies. Sections of UC were separated into vascular and Wharton's jelly (WJ) fractions, which were then digested individually to release MSC that were isolated by plastic adherence in a 10% fetal calf serum (FCS) medium, or a low serum medium designed for multipotent adult progenitor cells (MAPC). The resulting perivascular (PV) and WJ MSC lines were assayed for expression of characteristic markers and differentiation and immunosuppressive properties. MSC lines were readily derived from most UC tested. Cells grown in MAPC medium (MM) tended to be smaller and more elongated and expressed more nestin, but did not differ substantially in their growth rate, expression of other markers and differentiation capacity. All UC lines tested were adipogenic but poorly osteogenic, and were equivalent in their ability to suppress T-cell proliferation induced by phytohemagglutinin (PHA), activation beads and allostimulation. UC is a convenient, efficient source of MSC that can be expanded under low serum conditions for application on future studies of tissue regeneration and immunosuppression.

Babesia bovis: Effect of Albumax II and orotic acid in a low-serum in vitro culture

Bovine serum is an essential factor for the continuous in vitro growth of Babesia bovis parasites. Culture media typically contain 40% (v/v) of bovine serum. In the present study assays with low-serum media were performed. The growth of B. bovis Mo7 was successively lower in media with 30%, 20% and, principally, with 10% of serum. Without serum, the parasites were not able to propagate. In media with 10% of serum, the supplementation with Albumax™ II improved visibly the growth of B. bovis at the end of each cycle. Regarding the addition of orotic acid, no considerable effect was observed in media with 20% or 10% of serum, with or without Albumax™ II. B. bovis parasites cultured in vitro in all these media maintain their typical morphology during the intraerythrocytic stages.

Characterization of human skin-derived mesenchymal stem cell proliferation rate in different growth conditions.

This study investigated conditions for optimal in vitro propagation of human skin-derived mesenchymal stem cells (S-MSC). Forty primary skin-derived precursor cell (SKP) cultures were established from both male and female donors (age 29-65 years) and eight of them were randomly selected for in-depth characterization. Effects of basic fibroblast growth factor (FGF-2), epidermal growth factor (EGF), leukemia inhibiting factor (LIF) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) on S-MSC proliferation were investigated. Primary SKP cultures were >95% homogenous for CD90, CD73, and CD105 marker expression enabling to classify these cells as S-MSC. FGF-2 dose-dependent stimulation was observed in low serum medium only, whereas EGF neither stimulated S-MSC proliferation nor potentates the effect of FGF-2. Pronounced donor to donor differences among S-MSC cultures were observed in 3-day proliferation assay. This study demonstrates that homogenous S-MSC populations can be reproducibly isolated from individual donors of different age. Optimal cell culture conditions for in vitro propagation of S-MSC are B27 supplemented or low serum media with FGF-2 (4 ng/ml). EGF and LIF as well as db-cAMP are dispensable for S-MSC proliferation.

Multiple Mechanisms Are Responsible for Transactivation of the Epidermal Growth Factor Receptor in Mammary Epithelial Cells

The number of distinct signaling pathways that can transactivate the epidermal growth factor receptor (EGFR) in a single cell type is unclear. Using a single strain of human mammary epithelial cells, we found that a wide variety of agonists, such as lysophosphatidic acid (LPA), uridine triphosphate, growth hormone, vascular endothelial growth factor, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha, require EGFR activity to induce ERK phosphorylation. In contrast, hepatocyte growth factor can stimulate ERK phosphorylation independent of the EGFR. EGFR transactivation also correlated with an increase in cell proliferation and could be inhibited with metalloprotease inhibitors. However, there were significant differences with respect to transactivation kinetics and sensitivity to different inhibitors. In particular, IGF-1 displayed relatively slow transactivation kinetics and was resistant to inhibition by the selective ADAM-17 inhibitor WAY-022 compared with LPA-induced transactivation. Studies using anti-ligand antibodies showed that IGF-1 transactivation required amphiregulin production, whereas LPA was dependent on multiple ligands. Direct measurement of ligand shedding confirmed that LPA treatment stimulated shedding of multiple EGFR ligands, but paradoxically, IGF-1 had little effect on the shedding rate of any ligand, including amphiregulin. Instead, IGF-1 appeared to work by enhancing EGFR activation of Ras in response to constitutively produced amphiregulin. This enhancement of EGFR signaling was independent of both receptor phosphorylation and PI-3-kinase activity, suggestive of a novel mechanism. Our studies demonstrate that within a single cell type, the EGFR autocrine system can couple multiple signaling pathways to ERK activation and that this modulation of EGFR autocrine signaling can be accomplished at multiple regulatory steps.

The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway

The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.

Cotylenin A, a new differentiation inducer, and rapamycin cooperatively inhibit growth of cancer cells through induction of cyclin G2

Cotylenin A, a plant growth regulator, and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), are potent inducers of differentiation of myeloid leukemia cells. Recently, we found that cotylenin A and rapamycin effectively inhibited the proliferation of several human breast cancer cell lines including MCF-7. Herein, we demonstrate that cotylenin A and rapamycin rapidly and markedly induced the cyclin G2 gene expression in several cancer cells including MCF-7 cells. The growth arrest of the MCF-7 cells at the G1 phase, induced by the treatment with cotylenin A and rapamycin or the culture in low serum medium, markedly induced the cyclin G2 gene expression. Anticancer drugs including doxorubicin, etoposide and 5-fluorouracil also induced cyclin G2 expression during induction of growth arrest of the MCF-7 cell at the G1 phase or G2/M phase. Ectopically inducible cyclin G2 expression potently inhibited the proliferation of MCF-7 cells. Furthermore, cyclin G2 knockdown induced by cyclin G2 small interfering RNA markedly reduced the potency of cotylenin A plus rapamycin to induce growth inhibition. Taken together, our results suggest that cotylenin A and rapamycin induce inhibition of cancer cell growth through the induction of cyclin G2.

Tumorigenicity of primary mouse fibroblasts transformed by bovine papillomavirus type 1.

Cell lines transformed by bovine papillomavirus type 1 DNA were established from transfected primary fibroblast cultures of C57BL/6 mice. Southern blot analysis revealed that most of the viral DNA in the cells was in episomal oligomeric form. These cell lines were tested for tumorigenicity in nude mice in comparison to properties associated with malignant transformation including growth in low-serum medium, focus formation in agar, and resistance to natural killer cells. Two of the 5 cell lines were highly tumorigenic, producing spindle cell sarcomas in all animals. Metastatic spreading to local lymph nodes was also observed. The same cell lines also showed anchorage-independent growth in agar, but no correlation was observed between natural killer cell resistance and tumorigenicity. Viral transcripts were detected in two nontumorigenic and in two tumorigenic cell lines.

S6K1 Phosphorylates and Regulates Fragile X Mental Retardation Protein (FMRP) with the Neuronal Protein Synthesis-dependent Mammalian Target of Rapamycin (mTOR) Signaling Cascade

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein-synthesis-dependent synaptic plasticity.

Expression of neuronal proteins in cells from normal adult rat brain propagated in serial culture.

Cells have been cultured from the brains of 60-day-old rats and propagated through 12 passages. The cells contain the high and middle, but not low, molecular weight neurofilament subunits and neuron-specific enolase, demonstrated by immunoblotting and immunocytochemistry with redundant antibodies. The cells did not have the morphology of neurons when cultured in medium containing fetal calf serum and growth factors. In low serum medium containing the same growth factors with the addition of dibutyryl cyclic AMP, the cells became smaller and developed long processes. Three clonal lines derived from these cultures had the same properties. These observations are in agreement with recent observations using mouse and human brain tissue and demonstrate that proteins normally associated with neurons can be found in dividing cells cultured from the brains of young adult rats.

Starvation-dependent differential stress resistance protects normal but not cancer cells against high-dose chemotherapy

Strategies to treat cancer have focused primarily on the killing of tumor cells. Here, we describe a differential stress resistance (DSR) method that focuses instead on protecting the organism but not cancer cells against chemotherapy. Short-term starved S. cerevisiae or cells lacking proto-oncogene homologs were up to 1,000 times better protected against oxidative stress or chemotherapy drugs than cells expressing the oncogene homolog Ras2(val19). Low-glucose or low-serum media also protected primary glial cells but not six different rat and human glioma and neuroblastoma cancer cell lines against hydrogen peroxide or the chemotherapy drug/pro-oxidant cyclophosphamide. Finally, short-term starvation provided complete protection to mice but not to injected neuroblastoma cells against a high dose of the chemotherapy drug/pro-oxidant etoposide. These studies describe a starvation-based DSR strategy to enhance the efficacy of chemotherapy and suggest that specific agents among those that promote oxidative stress and DNA damage have the potential to maximize the differential toxicity to normal and cancer cells.

Genetic and epigenetic inactivation of T‐cadherin in human hepatocellular carcinoma cells

T-cadherin is an atypical cadherin and growing evidence has indicated that T-cadherin exerts tumor-suppressive effects on cancers of epithelial cell type and also causes positive effects on tumor angiogenesis. Human hepatocellular carcinoma (HCC) is a hypervascular tumor and T-cadherin has been shown to be overexpressed in intratumoral endothelial cells of HCCs. However, the expression status and functions of T-cadherin in hepatocytes or HCC cells remain unclear. Here, we demonstrated that T-cadherin was underexpressed in HCC cells (26.5%, 13/49 cases), but was frequently (77.6%, 38/49) overexpressed in intratumoral endothelial cells immunohistochemically. Semiquantitative RT-PCR analysis also showed that the T-cadherin gene was underexpressed in 7 of 11 HCC cell lines. Loss of heterozygosity analysis revealed that 32-38% of the 42 human HCC samples had allelic losses at this locus. Upon pharmacological treatment with demethylating agent 5-aza-2'-deoxycytidine or histone deacetylase inhibitor trichostatin A, T-cadherin promoter hypermethylation and/or histone deacetylation was frequently observed in HCC samples and cell lines. Functionally, enforced expression of T-cadherin induced G(2)/M cell cycle arrest, reduced cell proliferation in low serum medium, suppressed anchorage-independent growth in soft agar and increased sensitivity to TNFalpha-mediated apoptosis in HCC cells. Intriguingly, we found that T-cadherin significantly suppressed the activity of c-Jun, a crucial oncoprotein constitutively activated in HCC cells. To conclude, T-cadherin was differentially expressed in human HCCs. The underexpression of T-cadherin in HCC cells suggests it may be another critical event in addition to T-cadherin-mediated angiogenesis during HCC development.

Expansion and characterization of adipose tissue-derived stromal cells cultured with low serum medium

Adipose tissue contains a population of cells that have extensive self-renewal capacity and the ability to differentiate along multiple lineages. In addition, adipose tissue-derived stromal cells (ATSCs) are able to differentiate into various cell types that may be useful for autologous cell transplantation for defects of bone, cartilage, adipose, and tendon, etc. Most protocols for in vitro cultures of ATSCs include fetal bovine serum (FBS) as a nutritional supplement. However, in some cell cultures, it involves multiple doses of FBS, which raises a concern over possible infections as well as immunological reactions that are caused by medium-derived FBS proteins, sialic acid, etc. In this study, we were able to expand mouse ATSCs using low mouse serum media containing collagen type I, heparin-carrying polystyrene, and fibroblast growth factor (FGF)-2. These expanded mouse ATSCs maintained their multilineage potential for differentiation into adipocytes, osteoblasts, and chondrocytes. Therefore, this method, which uses autologous cells and low serum media, may be able to be utilized for clinical cell therapies.

Glial Cell Line-derived Neurotrophic Factor Reverses Ethanol-mediated Increases in Tyrosine Hydroxylase Immunoreactivity via Altering the Activity of Heat Shock Protein 90

We previously found that glial cell line-derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA) negatively regulates alcohol drinking (He, D. Y., McGough, N. N., Ravindranathan, A., Jeanblanc, J., Logrip, M. L., Phamluong, K., Janak, P. H., and Ron, D. (2005) J. Neurosci. 25, 619-628). Several studies suggest a role for GDNF in the regulation of tyrosine hydroxylase (TH) levels in the midbrain (Georgievska, B., Kirik, D., and Bjorklund, A. (2004) J. Neurosci. 24, 6437-6445). Up-regulation of TH levels has been reported as a hallmark of biochemical adaptations to in vivo chronic exposure to drugs of abuse, including ethanol (Ortiz, J., Fitzgerald, L. W., Charlton, M., Lane, S., Trevisan, L., Guitart, X., Shoemaker, W., Duman, R. S., and Nestler, E. J. (1995) Synapse 21, 289-298). We hypothesized that GDNF plays an important role in regulating prolonged ethanol-mediated increases in TH protein levels. Using the SH-SY5Y dopaminergic-like cell line, we found that the increase in TH levels in the presence of ethanol required the activation of the cAMP/PKA pathway and was reversed by GDNF. Ethanol treatment did not alter the mRNA level or protein translation of TH, but enhanced the stability of the protein that was decreased by GDNF. Interestingly, we observed that ethanol treatment resulted in an increase in TH association with the chaperone heat shock protein (HSP90) that was mediated by the cAMP/PKA pathway and inhibited by GDNF. Taken together, these data suggest that prolonged ethanol exposure leads to increased association of TH and HSP90 via the cAMP/PKA pathway, resulting in the stabilization and subsequent accumulation of TH. GDNF reverses this ethanol-mediated adaptation by inhibiting the interaction of TH with HSP90.

Live Yeast Cell Derivative Induces c-fos Expression in THP-1 Monocytes

Live Yeast Cell Derivative is a medicinal extract of Saccharomyces cerevisiae that has demonstrated efficacy in improving the rate and quality of wound healing in mouse and human systems. However, the mechanisms by which LYCD promotes healing are largely uncharacterized. In this report, we demonstrate that LYCD has effects on the transcriptional profile of the human monocytic cell line THP-1. Thirty minute exposures of THP-1 cells with LYCD induced a 6 to 44-fold, dose-dependent increase in the relative expression of the proto-oncogene c-fos in complete media containing 10% FBS or in low serum media containing 0.1% FBS. Furthermore, protein levels of c-Fos rise at 30 minutes of LYCD exposure and remained detectable for at least 120 minutes of LYCD exposure. However, the relative abundance of the c-fos transcript returned to basal levels by 120 minutes. LYCD also induced expression of c-jun with maximal expression of 3-fold at 60 minutes of exposure. Pretreatments with EGFR kinase inhibitor AG-1478 and the MEK1 inhibitor PD98059 blocked the LYCD-dependent increases in c-fos expression. Consistent with signaling through the EGFR, we have demonstrated by RT-PCR the presence of the mRNA for the EGFR (ErbB1/HER1) in THP-1 cells. Taken together these data suggest that LYCD acts through an EGFR-like cell surface receptor resulting in the activation of the EGFR kinase and the ERK1/2 signaling cascade.

Acyl‐CoA:cholesterol acyl transferase (ACAT1) is highly expressed in human breast cancer cell lines and ACAT inhibition reduces proliferation

We observed lipid droplets and an increase in cell cholesterol in breast cancer cells treated with fatty acids. We tested these cells for the expression of ACAT1, the enzyme that makes cholesterol esters (CE) from cholesterol and long-chain fatty acids. We found ACAT1 to be highly expressed at both the protein and mRNA levels in the highly proliferative MCF-10A and the invasive MDA-MB-231 breast cancer cell lines. ACAT1 expression was much lower in the estrogen receptor positive (ER+) breast cancer cell line MCF-7 and normal mammary epithelial cells. These data are consistent with existent array data showing increased expression of the ACAT1 gene soat1 in breast cancer compared to normal breast, lower expression in ER+ vs. ER- breast cancer, and co-expression of soat1 with breast cancer proliferation genes. The expression of ACAT1 correlated with CE content and total cell cholesterol. We hypothesized that high ACAT1 expression confers an advantage to proliferating cells when access to lipids needed for growth is limited. To test this hypothesis, we used the ACAT inhibitor CP-113,818. The inhibitor reduced proliferation of the high ACAT1-expressing cells and this effect was more pronounced in low serum media. When LDL was added to the media, it reduced the effect of ACAT inhibition. This suggests that ACAT1 has a role in facilitating growth and may confer a survival advantage to cancer cells when lipids are limited.