S3 Cells Research Articles

DR396, an apoptotic DNase γ inhibitor, attenuates high mobility group box 1 release from apoptotic cells

High mobility group box1 (HMGB1) is a non-histone chromatin chromosomal protein playing an important role in chromatin architecture and transcriptional regulation. Recently, HMGB1 has been shown to be secreted into extracellular milieu in necrosis and apoptosis, and involved in inflammatory responses. However, the mechanism by which apoptotic cells release HMGB1 is unclear. In this study, to investigate the mechanism of HMGB1 release, we searched inhibitors of HMGB1 release from apoptotic cells. As a result, three compounds, 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), Pontacyl Violet 6R (PV6R), and Fmoc-D-Cha-OH (FDCO) in our in-house chemical library were found to inhibit HMGB1 release from staurosporine (STS)-induced apoptotic HeLa S3 cells. Interestingly, these three compounds have been previously categorized into apoptotic DNase γ inhibitors. Therefore, we examined whether apoptotic nucleosomal DNA fragmentation is involved in the release of HMGB1 during apoptosis. Expectedly, DR396, which is the most potent and specific inhibitor of DNase γ, was found to almost completely inhibit both HMGB1 release and internucleosomal DNA cleavage in HeLa S3 cells transfected with DNase γ expression vector and stably expressing DNase γ (HeLa S3/γ cells). These results clearly suggest that nucleosomal DNA fragmentation catalyzed by DNase γ is critical in the release of HMGB1 from apoptotic cells.

The release of high mobility group box 1 in apoptosis is triggered by nucleosomal DNA fragmentation

High mobility group box 1 (HMGB1) initially identified as a non-histone chromosomal protein, which mainly functions as chromatin structure and transcriptional regulation, has been recently reported to be secreted into extracellular milieu in necrosis and apoptosis, and act as a proinflammatory mediator. However, the mechanism by which apoptotic cells release HMGB1 is not clear. In this study, we found that staurosporine (apoptosis-inducer)-induced HMGB1 release was associated with nucleosomal DNA fragmentation catalyzed by caspase-activated DNase (CAD) in WEHI-231 cells. Importantly, this event was effectively attenuated by the treatment of a pan-caspase inhibitor, Z-VAD-fmk, and by the inhibition of CAD-mediated DNA fragmentation by the expression of caspase-resistant inhibitor of CAD (ICAD-CR). In WEHI-231/ICAD-CR and WEHI-231/Puro cells, DNase γ-catalyzed nucleosomal DNA fragmentation occurred by anti-IgM antibody treatment was critical for HMGB1 release. Furthermore, in DNase γ stably-expressing HeLa S3 cells (HeLa S3/γ), the release of HMGB1 accompanied with nucleosomal DNA fragmentation was more apparent than that in parental HeLa S3 cells in which DNA fragmentation was scarcely observed. Taken together, these date suggest that nucleosomal DNA fragmentation catalyzed by CAD or DNase γ plays a pivotal role in HMGB1 release.

Anti-angiogenic effect of triterpenoidal saponins from Polygala senega.

Senegasaponins [senegin II (1), senegin III (2), senegin IV (3), senegasaponin a (4), and senegasaponin b (5)] from Polygala senega were re-discovered as selective anti-proliferative substances against human umbilical vein endothelial cells (HUVECs). Senegasaponins (1-5) showed anti-proliferative activity against HUVECs with IC(50) values in the range 0.6-6.2μM, and the selective index was 7-100-fold in comparison with those for several cancer cell lines, while the desacyl mixture of senegasaponins (6) and tenuifolin (7) lost anti-proliferative activity, indicating that the 28-O-glycoside moiety and methoxycinnamoyl group were essential for the HUVEC-selective growth inhibition of senegasaponins. Senegin III (2) inhibited the vascular endothelial growth factor (VEGF)-induced in vitro tubular formation of HUVECs and basic fibroblast growth factor (bFGF)-induced in vivo neovascularization in the mouse Matrigel plug assay. Moreover, senegin III (2) suppressed tumor growth in the ddY mice s.c.-inoculated murine sarcoma S180 cells. The analysis of the action mechanism of senegin III (2) suggested that the induction of pigment epithelium-derived factor (PEDF) would contribute to the anti-angiogenic effects of senegasaponins.

Role of Autophagy in Sonodynamic Therapy-Induced Cytotoxicity in S180 Cells

Few reports have been published on the potential role of autophagy in the efficacy of sonodynamic therapy (SDT). This study was to determine whether autophagy occurred after SDT and to investigate its relationship with apoptosis by performing inhibitor studies. In vitro murine sarcoma 180 (S180) cells were examined at different time points following SDT. Transmission electron microscopy (TEM) was used to identify the formation of autophagosomes. Western blots were used to assess the processing of LC3-I to LC3-II. Confocal microscopy was performed to reveal co-localization between mitochondria and autophagic vacuoles and re-distribution of apoptosis related proteins after sono-damage. Inhibitors of apoptosis and autophagy were used to determine the contributions of the two cellular responses to SDT efficacy. Autophagy was indentified by TEM observation of the presence of double-membrane delineated autophagic vesicles and by immunoblot observation of the increased LC3-II levels. The autophagy inhibitors, both 3-methyladenine (3-MA) and Bafilomycin A1 (Ba A1), were found to significantly enhance SDT-induced cell death. Blocking autophagy also led to increased dissipation of mitochondria potential, caspase-3 activity and the ultimate cell apoptosis. Whereas the pan-caspase inhibitor, z-VAD-fmk partially prevented SDT-induced cytotoxicity but did not obviously improve the autophagic vacuolization and mitochondria depolarization. This study suggests for the first time that autophagy participate in SDT-induced cell death and combination of SDT with autophagy inhibitors, especially preventing autophagy at the early stage by 3-MA, can significantly enhance the anti-tumor effect of SDT through induction of apoptosis and necrosis. (E-mail: lshaof@snnu.edu.cn)

Synthesis of isoxazoline-linked chlorins and their in vitro cell viabilities

A concise synthesis of isoxazoline-linked chlorins is described. This approach is carried out from methyl pyropheophorbide-a as the starting material via 1,3-dipolar cycloaddition of a vinyl group on the periphery with nitrile oxide to give regioselective products with excellent yields. This method represents an extensive and efficient entry into the functionalization of chlorins with a chlorophyll-α skeleton. Moreover, we have examined a preliminary in vitro effect of these new derivatives on mouse sarcoma S-180 cell line in photodynamic therapy.

The complete preventive effect of homologous tumor vaccines – Based on a 5-year experimental study in mice

By using a frozen-thaw method, we developed homologous and heterologous cell-based tumor vaccines, which were derived from mouse H22 hepatoma, S180 sarcoma and human A549 lung carcinoma, SK-OV-3 ovarian, and SMMC-7721 hepatoma cell lines. The prophylactic and therapeutic effects of those vaccines were evaluated in mice challenged with live H22 or S180 cells. The result demonstrated that homologous vaccines and heterologous vaccines had no therapeutic effect on tumor growth. However, homologous vaccines showed a complete prevention against live H22 and S180 cell challenge and they could stimulate cross-immune response of anti-tumor in mice. Furthermore, these tumor-free mice immunized with homologous vaccines showed full protection against the repeat challenge every 3 months for 5 years. The study also revealed that tumor-free female, not male, mice transferred anti-tumor ability to some of their offsprings. Heterologous vaccines exhibited no protective effect on tumor development. Immunological analysis discovered that activities of CTLs and NK were enhanced and the levels of IL-2, IL-12 and IFN-γ were significantly increased. Our results demonstrated that homologous tumor vaccines could elicit complete cross-protection against the lethal challenge of tumor cells through enhancing cell-mediated immune response, which lasted for 5 years in mice. These observations may provide a new vaccine strategy for tumor prevention.

GLIS, a bioactive proteoglycan fraction from Ganoderma lucidum, displays anti-tumour activity by increasing both humoral and cellular immune response

Aims Ganoderma lucidum, a traditional Chinese medicine, is well known as a modulator of functions of the immune system as well as an anti-tumour agent. However, its active compounds and their molecular mechanisms of action are not well established. GLIS, a proteoglycan isolated from the fruiting body of G. lucidum, stimulates directly the activation of B lymphocytes. In this work, the immunoactivation capacities of GLIS as well as its anti-tumour effect were investigated in vitro and in vivo. Main methods Tumour-bearing mice were prepared by inoculation of mouse sarcoma S180 cells into BALB/c mice. Lymphocytes and bone marrow-derived macrophages were isolated from spleen and tibia/femurs, respectively. After stimulation with GLIS different immune responses of these cells were analysed. Anti-tumour effect of GLIS was determined. Key findings After treatment with GLIS, spleen-derived B lymphocytes from tumour-bearing mice became activated, proliferated and produced large amounts of immunoglobulins. Bone marrow-derived macrophages from tumour-bearing mice also became activated after exposure to GLIS, and they produced important immunomodulatory substances, such as IL-1β, TNF-α and reactive nitrogen intermediates, like NO. GLIS markedly increased phagocytosis of macrophages, and very importantly, it markedly raised the macrophage-mediated tumour cytotoxicity. Treatment of mice with GLIS caused an inhibition of mouse sarcoma S180 tumour growth by 60% in vivo. Significance These results indicate that GLIS exhibits a capacity to increase remarkably both humoral and cellular immune activities of tumour-bearing mice and inhibits tumour growth significantly. The anti-tumour effect of GLIS results from its capacity to increase the host's immune activity.

왕겨에 의한 신령버섯균사체 액체배양액의 생쥐 항복수암성 증가

The effects of rice hull (RH) powder on the anticarcinogenic activity of submerged-liquid cultures of Agaricus blazei Murill (AB) were assessed for mouse ascites cancers induced by mouse Sarcoma S-180 (S-180) cancer cells. Optimal growth of AB mycelia in the basal liquid culture medium, containing soybean meal, was achieved by culturing at for 5 days, when evaluated by -glucan content, Brix, and mycelial weight, relative to other culture conditions. Hot-water extract (HWE) of the submergedliquid culture of AB mycelia grown at for 5 days exhibited a stronger anticarcinogenic activity, relative to HWE from other culture conditions. No such effects were obtained from AB mycelial cultures by alternative temperature-controlling cultures. Both cytotoxicity for S-180 cells and anticarcinogenic potentials for mouse ascites cancer of the HWE from AB mycelia grown in the basal medium containing 1% RH powder for 5 days at were significantly (p for 5 days, enhanced anticarcinogenic activity against S-180 cell-induced mouse ascites cancer, and suggest that RH powder is an excellent ingredient for the improvement of the anticarcinogenic potentials of the submerged-liquid culture of mushroom mycelia.

Hypoxia‐Selective Growth Inhibition of Cancer Cells by Furospinosulin‐1, a Furanosesterterpene Isolated from an Indonesian Marine Sponge

It is generally accepted that cancer cells, which have adapted to the hypoxic environments in tumor tissues, aggravate cancer pathology by promoting tumor growth, angiogenesis, metastasis, and drug resistance. Therefore, compounds that selectively inhibit the growth of tumor cells in hypoxic environments are expected to provide new leads for promising anticancer drugs. Furospinosulin-1, a marine-sponge-derived furanosesterterpene, exhibited selective antiproliferative activity against DU145 human prostate cancer cells under hypoxic conditions in concentrations ranging from 1 to 100 μM. Furospinosulin-1 also demonstrated antitumor activity at 10-50 mg kg(-1) oral administration in a mouse model inoculated with sarcoma S180 cells. Mechanistic analysis revealed that furospinosulin-1 suppresses transcription of the insulin-like growth factor-2 gene (IGF-2), which is selectively induced under hypoxic conditions through prevention of the binding of nuclear proteins to the Sp1 consensus sequence in the IGF-2 promoter region.

Signal Transducers and Activators of Transcription-1 (STAT1) Regulates microRNA Transcription in Interferon γ-Stimulated HeLa Cells

BackgroundConstructing and modeling the gene regulatory network is one of the central themes of systems biology. With the growing understanding of the mechanism of microRNA biogenesis and its biological function, establishing a microRNA-mediated gene regulatory network is not only desirable but also achievable.MethodologyIn this study, we propose a bioinformatics strategy to construct the microRNA-mediated regulatory network using genome-wide binding patterns of transcription factor(s) and RNA polymerase II (RPol II), derived using chromatin immunoprecipitation following next generation sequencing (ChIP-seq) technology. Our strategy includes three key steps, identification of transcription start sites and promoter regions of primary microRNA transcripts using RPol II binding patterns, selection of cooperating transcription factors that collaboratively function with the transcription factors targeted by ChIP-seq assay, and construction of the network that contains regulatory cascades of both transcription factors and microRNAs.Principal FindingsUsing CAMDA (Critical Assessment of Massive Data Analysis) 2009 data set that includes ChIP-seq data on RPol II and STAT1 (signal transducers and activators of transcription 1) in HeLa S3 cells in control condition and with interferon γ stimulation, we first identified promoter regions of 83 microRNAs in HeLa cells. We then identified two potential STAT1 collaborating factors, AP-1 and C/EBP (CCAAT enhancer-binding proteins), and further established eight feedback network elements that may regulate cellular response during interferon γ stimulation.ConclusionsThis study offers a bioinformatics strategy to provide testable hypotheses on the mechanisms of microRNA-mediated transcriptional regulation, based upon genome-wide protein-DNA interaction data derived from ChIP-seq experiments.

K11-Linked Polyubiquitination in Cell Cycle Control Revealed by a K11 Linkage-Specific Antibody

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

Anticancer effect of polysaccharide isolated from Polyporus sp. M05.

Objective To study effect and mechanism of fungus polysaccharide PSM-a of Polyporus sp. M05 on S180 bearing mice. Methods MTT method was used to detect the inhibiting role of PSM-a on S180 cells proliferation in vitro. S180 mice model was established, and was administered by gavage. Tumor volume was detected, and the ratio of tumor to mile weight and inhibiting tumor rate. The activity of NK and LAK cells on the target cells was analyzed by MTT colorimetric assay; HE stain was used to detect the necrosis of tumor cells. Restilts PSM-a could inhibit S180 cells grouth in vitro. PSM-a could decrease the tumor weight and increase the ratio of tumor volume and mice weight; Tumor inhibiting rate reached 80% and above when treated with 250μg/ml PSM-a. PSM-a could increase the activity of NK and LAK cells, and necrosis happened. Conclusion PSM-a could significantly inhibit the growth of S180 cells, and the mechanism bnay be related with the increased killing activity of immunne cells to tumor cells. Key words: Polyporaceae; polysaccharides; Neoplasms

Medium osmolarity-dependent biosynthesis of renal cellular sulfoglycolipids is mediated by the MAPK signaling pathway

Verots S3 and Vero317 cells were shown by metabolic labeling with 35S-sulfate to contain many more sulfoglycosphingolipids than original Vero cells derived from African green monkey kidney. The activity of galactosyl ceramide sulfotransferase (GST) was shown to be 89- and 92-fold higher in Vero317 cells and Verots S3 cells, respectively, than that of the parent cells, whereas the activity of the degradation enzyme, arylsulfatase A, was unchanged among all the three cell strains. GST gene transcript levels in Verots cells were 14.3-fold higher than those in Vero cells. The cell adhesiveness to the culture plate under hypertonic stress was strengthened significantly in both mutant strains. Among the major sulfoglycolipids of the Verots S3 cell line, assigned as SM4s, SM3, SM2a, and SB1a, the incorporation of 35S-sulfate into SM3, SM2a and SB1a was upregulated with the increasing tonicity of the medium. Sulfoglycolipids in these renal cells seemed to contribute to the membrane barrier against hypertonic media as shown previously in another renal cell line, MDCK (Niimura and Nagai, 2008). Sulfoglycolipid synthesis was suppressed with the p38 (MAPK) inhibitor SB203580 and/or with the MEK-1/2 (MAPKK) inhibitor PD98059, and with the tyrosine kinase inhibitor genistein, which also reduced the sulfoglycolipid synthesis in a dose-dependent manner. Further the administration of the MAPK/MAPKK inhibitors to the culture medium reduced significantly the viability of Verots S3 cells under hypertonic stress. These findings suggest that sulfoglycolipid synthesis in those renal cells may be regulated to adapt to the renal osmotic circumstances by the medium's osmolarity via the MAPK signaling pathway.

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G(2)/M transition. We find that activation of Aurora A first occurs at centrosomes at late G(2) and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.

The ruthenium complex cis-(dichloro)tetrammineruthenium(III) chloride induces apoptosis and damages DNA in murine sarcoma 180 cells

Ruthenium (III) complexes are increasingly attracting the interest of researchers due to their promising pharmacological properties. Recently, we reported that the cis-(dichloro)tetrammineruthenium (III) chloride compound has cytotoxic effects on murine sarcoma 180 (S-180) cells. In an effort to understand the mechanism responsible for their cytotoxicity, study we investigated the genotoxicity, cell cycle distribution and induction of apoptosis caused by cis- (dichloro) tetrammineruthenium (III) chloride in S-180 tumour cells. cis-(dichloro) tetrammineruthenium (III) chloride treatment induced significant DNA damage in S-180 cells, as detected by the alkaline comet assay. In the cell cycle analysis, cis-(dichloro) tetrammineruthenium (III) chloride caused an increase in the number of cells in G1 phase, accompanied by a decrease in the S and G2 phases after 24 h of treatment. In contrast, the cell cycle distribution of S-180 cells treated with cis-(dichloro) tetrammineruthenium (III) chloride for 48 h showed a concentration-dependent increase in the sub-G1 phase (indicating apoptosis), with a corresponding decrease in cells in the G1, S and G2 phases. In addition, cis-(dichloro) tetrammineruthenium(III) chloride treatment induced apoptosis in a time-dependent manner,as observed by the increased numbers of annexin V-positive cells. Taken together, these findings strongly demonstrate that DNA damage, cell cycle changes and apoptosis may correlate with the cytotoxic effects of cis-(dichloro) tetrammineruthenium (III) chloride on S-180 cells.

Development of cell-penetrating peptide-modified MPEG-PCL diblock copolymeric nanoparticles for systemic gene delivery

To develop a safe and efficient systemic non-viral gene vector, methoxy poly(ethylene glycol) (MPEG)/poly(ɛ-caprolactone) (PCL) diblock copolymers conjugated with a Tat analog through the ester or disulfide linkage were synthesized and their suitability as a systemic non-viral gene carrier evaluated. The physicochemical properties of the MPEG-PCL diblock copolymers were determined by GPC, 1H NMR and FT-IR spectroscopy. The particle sizes and in vitro (COS7 and S-180 cells) transfection efficiencies and cytotoxicity were evaluated. Furthermore, the luciferase activity was then determined in various tissues after intravenous injection of MPEG-PCL-SS-Tat/pCMV-Luc complex into mice bearing S-180 cells. The particle sizes of the MPEG-PCL-Tat copolymers with or without pDNA were about 40 and 60 nm, respectively. The luciferase activity in COS7 cells transfected with pCMV-Luc with MPEG-PCL-ester-Tat or MPEG-PCL-SS-Tat was higher than that with pDNA only. MPEG-PCL-SS-Tat greatly increased the transfection efficiency compared to MPEG-PCL-ester-Tat in COS7 and S-180 cells. In an in vitro cytotoxicity test MPEG-PCL-SS-Tat did not induce any remarkable cytotoxicity. In an in vivo experiment, the synthesized MPEG-PCL-SS-Tat copolymers promoted the delivery and expression of pDNA into tumor tissue in tumor-bearing mice. In conclusion, this vector might be applicable as a tumor-targeting non-viral systemic gene carrier in the clinical setting.

Strain-Specific Susceptibility for Pulmonary Metastasis of Sarcoma 180 Cells in Inbred Mice

Most cancer deaths are a result of metastasis. To extend our understanding of the factors that influence the process, we aimed to develop a mouse model of pulmonary metastasis that can be assayed in multiple inbred mouse strains for further use in identification of host genetic variants that influence metastasis. We used i.v. injection of Sarcoma 180 (S180) cells, which can be tracked and quantified by bioluminescence imaging. We observed growth of S180 cells solely in the lung and observed a wide range of pulmonary metastasis among inbred mouse strains. Interestingly, we noted that the BTBRT+tf/J strain exhibited complete clearance and provide evidence that the mechanism of resistance may involve immune factors, as strains subjected to whole-body irradiation are significantly more susceptible to tumor growth. One possible mechanism of resistance to pulmonary metastasis in BTBRT+tf/J mice may require T-cell function. Our experiments present a new mouse model for further characterization of the genetics and mechanisms of pulmonary metastasis.

Enhanced immunostimulatory and antitumor activity of different derivatives of κ-carrageenan oligosaccharides from Kappaphycus striatum

Chemical modification of carbohydrates can lead to differences in their biological activities. We previously showed that kappa-carrageenan oligosaccharides from Kappaphycus striatum have antitumor and immunomodulation effects on S180-bearing mice. In this study, we tested the hypothesis that different chemical modifications of carrageenan oligosaccharides enhance their activities. The mice inoculated with S180 cell suspension were treated p.o. with carrageenan oligosaccharides and their sulfated, acetylated, and phosphorylated derivatives (50, 100, and 200 mu g g(-1)) for 14 days. Transplantable tumor inhibition rate and macrophage phagocytosis, quantitative hemolysis of sheep red blood cells, lymphocyte proliferation, the activity of natural killer cells, production of interleukin-2, and tumor necrosis factor-alpha were also analyzed. As expected, treatment with different kappa-carrageenan oligosaccharides derivatives resulted in an increase in tumor inhibition rate and macrophage phagocytosis and cellular immunity, especially on spleen lymphocyte proliferation. The sulfated derivative at the dose 200 mu g g(-1) per day showed the highest antitumor activity with the 54.12% tumor weight inhibition and elicited an increase in nature killer cells activity up to 76.1% on S180-bearing mice, which were both significantly higher than the unmodified oligosaccharides. It suggested that chemical modification (especially sulfation) of carrageenan oligosaccharides can enhance their antitumor effect and boost their antitumor immunity.

Evaluation of gene expression signatures induced by stromal factors as a predictor of survival in breast cancer.

10607 Background: Recent findings present the tumor microenvironment as an innovative therapeutic target in contrast to traditional tumor cell directed therapy. Fibroblasts have been identified to play an important role in breast cancer progression. We analyzed the effect of normal mammary fibroblasts on gene expression in human breast epithelial cells in a 3D coculture and it's correlation to clinical data. Methods: Nonmalignant HMT3522 S1 human breast epithelial cells were cultured in 3D collagen gels as mono- or coculture with normal human mammary fibroblasts. Gene expression profiles of S1 cells from co- and monoculture were analyzed using the Affymetrix HG-U133 Plus 2.0 GeneChip. Signal values were calculated by means of RMA using R Bioconductor 2.4, then log2 transformed. Transcripts with a log ratio ≥ ± 1.5 were defined as differentially expressed. Using published microarray profiles of 295 human breast tumors with associated clinical data t- tests, hierarchical cluster and Kaplan-Meier analysis were performed to stratify the patients according to the generated gene expression profiles. Results: S1 cells in 3D coculture differentiated into polarized acinus like structures and underwent growth arrest after 10 d of culture, whereas S1 cells in 3D monoculture showed unorganized and progressive growth. 99 transcripts were upregulated in S1 cells from co-compared to monoculture. Known functions of these genes mainly relate to extracellular matrix constitution and differentiation. 36 transcripts were upregulated in S1 cells from monoculture, predominantly involved in cell cycle regulation. Hierarchical cluster analysis stratified the patients into poor and good prognosis groups. Further on, Kaplan-Meier analysis showed that 21 selected genes were highly informative in identifying patients with poor or good outcome (p < 0.01). Conclusions: Gene expression changes in human breast epithelial cells induced by normal mammary fibroblasts provide gene expression profiles that clearly stratify patients into prognostic groups. Our 3D culture model proves to be a valuable in vitro model for cancer research and emphasizes the relevance of the tumor microenvironment as a target for breast cancer therapy. No significant financial relationships to disclose.

Regulation of the creatine transporter by AMP-activated protein kinase in kidney epithelial cells

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na(+)-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten V(max) parameter. [(14)C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress.