Sterol-like drugs potentiate statin-triggered prostate cancer cell death by inhibiting SREBP2 nuclear translocation.

There is an urgent need to provide immediate and effective options for the treatment of prostate cancer (PCa) to prevent progression to lethal castration-resistant PCa (CRPC). The mevalonate (MVA) pathway is dysregulated in PCa, and statin drugs commonly prescribed for hypercholesterolemia, effectively target this pathway. Statins exhibit anti-PCa activity, however the resulting intracellular depletion of cholesterol triggers a feedback loop that restores MVA pathway activity, thus diminishing statin efficacy and contributing to resistance. To identify drugs that block this feedback response and enhance the pro-apoptotic activity of statins, we performed a high-content image-based screen of a 1508 drug library, enriched for FDA-approved compounds. Two of the validated hits, Galeterone (GAL) and Quinestrol, share the cholesterol-related tetracyclic structure, which is also evident in the FDA-approved CRPC drug Abiraterone (ABI). Molecular modeling revealed that GAL, Quinestrol and ABI not only share structural similarity with 25-hydroxy-cholesterol (25HC) but were also predicted to bind similarly to a known protein-binding site of 25HC. This suggested GAL, Quinestrol and ABI are sterol-mimetics and thereby inhibit the statin-induced feedback response. Cell-based assays demonstrated that these agents inhibit nuclear translocation of sterol-regulatory element binding protein 2 (SREBP2) and the transcription of MVA genes. Sensitivity was independent of androgen status and the Fluva-GAL combination significantly impeded CRPC tumor xenograft growth. By identifying cholesterol-mimetic drugs that inhibit SREBP2 activation upon statin treatment, we provide a potent "one-two punch" against CRPC progression and pave the way for innovative therapeutic strategies to combat additional diseases whose etiology is associated with SREBP2 dysregulation.

The carboxyl-terminal sequence of PUMA binds to both anti-apoptotic proteins and membranes.

Anti-apoptotic proteins such as BCL-XL promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-XL, BCL-2, BCL-W, and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively 'double-bolt locks' the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins.

Computational pharmacogenomic screen identifies drugs that potentiate the anti-breast cancer activity of statins.

Statins, a family of FDA-approved cholesterol-lowering drugs that inhibit the rate-limiting enzyme of the mevalonate metabolic pathway, have demonstrated anticancer activity. Evidence shows that dipyridamole potentiates statin-induced cancer cell death by blocking a restorative feedback loop triggered by statin treatment. Leveraging this knowledge, we develop an integrative pharmacogenomics pipeline to identify compounds similar to dipyridamole at the level of drug structure, cell sensitivity and molecular perturbation. To overcome the complex polypharmacology of dipyridamole, we focus our pharmacogenomics pipeline on mevalonate pathway genes, which we name mevalonate drug-network fusion (MVA-DNF). We validate top-ranked compounds, nelfinavir and honokiol, and identify that low expression of the canonical epithelial cell marker, E-cadherin, is associated with statin-compound synergy. Analysis of remaining prioritized hits led to the validation of additional compounds, clotrimazole and vemurafenib. Thus, our computational pharmacogenomic approach identifies actionable compounds with pathway-specific activities.

Chemical Genetics Screen Identifies COPB2 Tool Compounds That Alters ER Stress Response and Induces RTK Dysregulation in Lung Cancer Cells.

Activating mutations in the epidermal growth factor receptor (EGFR) are common driver mutations in non-small cell lung cancer (NSCLC). First, second and third generation EGFR tyrosine kinase inhibitors (TKIs) are effective at inhibiting mutant EGFR NSCLC, however, acquired resistance is a major issue, leading to disease relapse. Here, we characterize a small molecule, EMI66, an analog of a small molecule which we previously identified to inhibit mutant EGFR signalling via a novel mechanism of action. We show that EMI66 attenuates receptor tyrosine kinase (RTK) expression and signalling and alters the electrophoretic mobility of Coatomer Protein Complex Beta 2 (COPB2) protein in mutant EGFR NSCLC cells. Moreover, we demonstrate that EMI66 can alter the subcellular localization of EGFR and COPB2 within the early secretory pathway. Furthermore, we find that COPB2 knockdown reduces the growth of mutant EGFR lung cancer cells, alters the post-translational processing of RTKs, and alters the endoplasmic reticulum (ER) stress response pathway. Lastly, we show that EMI66 treatment also alters the ER stress response pathway and inhibits the growth of mutant EGFR lung cancer cells and organoids. Our results demonstrate that targeting of COPB2 with EMI66 presents a viable approach to attenuate mutant EGFR signalling and growth in NSCLC.

A reference library for assigning protein subcellular localizations by image-based machine learning.

Confocal micrographs of EGFP fusion proteins localized at key cell organelles in murine and human cells were acquired for use as subcellular localization landmarks. For each of the respective 789,011 and 523,319 optically validated cell images, morphology and statistical features were measured. Machine learning algorithms using these features permit automated assignment of the localization of other proteins and dyes in both cell types with very high accuracy. Automated assignment of subcellular localizations for model tail-anchored proteins with randomly mutated C-terminal targeting sequences allowed the discovery of motifs responsible for targeting to mitochondria, endoplasmic reticulum, and the late secretory pathway. Analysis of directed mutants enabled refinement of these motifs and characterization of protein distributions in within cellular subcompartments.

Genome-wide analysis of Homo sapiens, Arabidopsis thaliana, and Saccharomyces cerevisiae reveals novel attributes of tail-anchored membrane proteins.

BACKGROUND: Tail-anchored membrane proteins (TAMPs) differ from other integral membrane proteins, because they contain a single transmembrane domain at the extreme carboxyl-terminus and are therefore obliged to target to membranes post-translationally. Although 3-5% of all transmembrane proteins are predicted to be TAMPs only a small number are well characterized. RESULTS: To identify novel putative TAMPs across different species, we used TAMPfinder software to identify 859, 657 and 119 putative TAMPs in human (Homo sapiens), plant (Arabidopsis thaliana), and yeast (Saccharomyces cerevisiae), respectively. Bioinformatics analyses of these putative TAMP sequences suggest that the list is highly enriched for authentic TAMPs. To experimentally validate the software predictions several human and plant proteins identified by TAMPfinder that were previously uncharacterized were expressed in cells and visualized at subcellular membranes by fluorescence microscopy and further analyzed by carbonate extraction or by bimolecular fluorescence complementation. With the exception of the pro-apoptotic protein harakiri, which is, peripherally bound to the membrane this subset of novel proteins behave like genuine TAMPs. Comprehensive bioinformatics analysis of the generated TAMP datasets revealed previously unappreciated common and species-specific features such as the unusual size distribution of and the propensity of TAMP proteins to be part of larger complexes. Additionally, novel features of the amino acid sequences that anchor TAMPs to membranes were also revealed. CONCLUSIONS: The findings in this study more than double the number of predicted annotated TAMPs and provide new insights into the common and species-specific features of TAMPs. Furthermore, the list of TAMPs and annotations provide a resource for further investigation.

Activated ErbB3 Translocates to the Nucleus via Clathrin-independent Endocytosis, Which Is Associated with Proliferating Cells.

Members of the receptor tyrosine kinase family (RTK) have been shown to be present in the nucleus of cells; however, the mechanisms underlying their trafficking to the nucleus, and their relevance once there are poorly understood. In the present study, we focus on the RTK ErbB3 and elucidate the mechanisms regulating its trafficking. We show that heregulin-stimulation induces trafficking of phosphorylated ErbB3 from the plasma membrane to the nucleus via a clathrin-independent mechanism. Nuclear import of ErbB3 occurs via importin ß1, which drives the receptor through the nuclear pore complex. In the nucleus, ErbB3 interacts with transcription complexes, and thereby has a role in transcriptional regulation. Our results also demonstrate that ErbB3 nuclear localization is transient as it is exported out of the nucleus by the nuclear receptor protein crm-1. Analysis of normal, regenerating tissues, and tumors showed that ErbB3 nuclear translocation is a common event in proliferating tissues.

Ep-CAM RNA expression predicts metastasis-free survival in three cohorts of untreated node-negative breast cancer.

Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention as a prognostic factor in breast cancer. We aimed to validate the influence of Ep-CAM RNA expression in untreated node-negative breast cancer. Ep-CAM RNA expression was evaluated utilizing microarray-based gene-expression profiling in 194 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. The prognostic significance of Ep-CAM RNA expression for disease-free survival (DFS), metastasis-free survival (MFS), and breast cancer-specific overall survival (OS) was evaluated in univariate and multivariate analysis adjusted for age, grading, pTstage, ER as well as PR receptor and HER-2 status. Additionally, Ep-CAM RNA expression was compared with immunohistochemistry (IHC) for Ep-CAM in 194 patients. The prognostic impact of Ep-CAM gene expression was validated in further 588 node-negative breast cancer patients. Levels of Ep-CAM RNA expression showed a significant correlation with IHC (P = 0.001) and predicted in univariate analysis DFS (P = 0.001, HR = 2.4), MFS (P = 0.003, HR = 2.5), and OS (P = 0.002, HR = 3.1) accurately. The prognostic influence of Ep-CAM RNA was significant also in multivariate analysis for DFS (P = 0.017, HR = 2.0), MFS (P = 0.049, HR = 1.9), and OS (P = 0.042, HR = 2.3), respectively. The association with MFS was confirmed in an independent validation cohort in univariate (P = 0.006, HR = 1.9) and multivariate (P = 0.035, HR = 1.7) analysis. Ep-CAM RNA correlated with the proliferation metagene (P < 0.001, R=0.425) Nevertheless, in multivariate analysis, Ep-CAM was associated with MFS independent from the proliferation metagene (P = 0.030, HR = 1.8). In conclusion, Ep-CAM RNA expression is associated with poor MFS in three cohorts of untreated node-negative breast cancer.

Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.

Transcription factors ETF, E2F, and SP-1 are involved in cytokine-independent proliferation of murine hepatocytes.

UNLABELLED: The cellular basis of liver regeneration has been intensely investigated for many years. However, the mechanisms initiating hepatocyte "plasticity" and priming for proliferation are not yet fully clear. We investigated alterations in gene expression patterns during the first 72 hours of C57BL/6N mouse hepatocyte culture on collagen monolayers (CM), which display a high basal frequency of proliferation in the absence of cytokines. Although many metabolic genes were down-regulated, genes related to mitogen-activated protein kinase (MAPK) signaling and cell cycle were up-regulated. The latter genes showed an overrepresentation of transcription factor binding sites (TFBS) for ETF (TEA domain family member 2), E2F1 (E2F transcription factor 1), and SP-1 (Sp1 transcription factor) (P < 0.001), all depending on MAPK signaling. Time-dependent increase of ERK1/2 phosphorylation occurred during the first 48 hours (and beyond) in the absence of cytokines, accompanied by an enhanced bromodeoxyuridine labeling index of 20%. The MEK inhibitor PD98059 blunted these effects indicating MAPK signaling as major trigger for this cytokine-independent proliferative response. In line with these in vitro findings, liver tissue of mice challenged with CCl(4) displayed hepatocytes with intense p-ERK1/2 staining and nuclear SP-1 and E2F1 expression. Furthermore, differentially expressed genes in mice after partial hepatectomy contained overrepresented TFBS for ETF, E2F1, and SP-1 and displayed increased expression of E2F1. CONCLUSION: Cultivation of murine hepatocytes on CM primes cells for proliferation through cytokine-independent activation of MAPK signaling. The transcription factors ETF, E2F1, and SP-1 seem to play a pronounced role in mediating proliferation-dependent differential gene expression. Similar events, but on a shorter time-scale, occur very early after liver damage in vivo.

Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer.

INTRODUCTION: The purpose of this work was to study the prognostic influence in breast cancer of thioredoxin reductase 1 (TXNRD1) and thioredoxin interacting protein (TXNIP), key players in oxidative stress control that are currently evaluated as possible therapeutic targets. METHODS: Analysis of the association of TXNRD1 and TXNIP RNA expression with the metastasis-free interval (MFI) was performed in 788 patients with node-negative breast cancer, consisting of three individual cohorts (Mainz, Rotterdam and Transbig). Correlation with metagenes and conventional clinical parameters (age, pT stage, grading, hormone and ERBB2 status) was explored. MCF-7 cells with a doxycycline-inducible expression of an oncogenic ERBB2 were used to investigate the influence of ERBB2 on TXNRD1 and TXNIP transcription. RESULTS: TXNRD1 was associated with worse MFI in the combined cohort (hazard ratio = 1.955; P < 0.001) as well as in all three individual cohorts. In contrast, TXNIP was associated with better prognosis (hazard ratio = 0.642; P < 0.001) and similar results were obtained in all three subcohorts. Interestingly, patients with ERBB2-status-positive tumors expressed higher levels of TXNRD1. Induction of ERBB2 in MCF-7 cells caused not only an immediate increase in TXNRD1 but also a strong decrease in TXNIP. A subsequent upregulation of TXNIP as cells undergo senescence was accompanied by a strong increase in levels of reactive oxygen species. CONCLUSIONS: TXNRD1 and TXNIP are associated with prognosis in breast cancer, and ERBB2 seems to be one of the factors shifting balances of both factors of the redox control system in a prognostic unfavorable manner.

Comparison of scores for bimodality of gene expression distributions and genome-wide evaluation of the prognostic relevance of high-scoring genes.

BACKGROUND: A major goal of the analysis of high-dimensional RNA expression data from tumor tissue is to identify prognostic signatures for discriminating patient subgroups. For this purpose genome-wide identification of bimodally expressed genes from gene array data is relevant because distinguishability of high and low expression groups is easier compared to genes with unimodal expression distributions.Recently, several methods for the identification of genes with bimodal distributions have been introduced. A straightforward approach is to cluster the expression values and score the distance between the two distributions. Other scores directly measure properties of the distribution. The kurtosis, e.g., measures divergence from a normal distribution. An alternative is the outlier-sum statistic that identifies genes with extremely high or low expression values in a subset of the samples. RESULTS: We compare and discuss scores for bimodality for expression data. For the genome-wide identification of bimodal genes we apply all scores to expression data from 194 patients with node-negative breast cancer. Further, we present the first comprehensive genome-wide evaluation of the prognostic relevance of bimodal genes. We first rank genes according to bimodality scores and define two patient subgroups based on expression values. Then we assess the prognostic significance of the top ranking bimodal genes by comparing the survival functions of the two patient subgroups. We also evaluate the global association between the bimodal shape of expression distributions and survival times with an enrichment type analysis.Various cluster-based methods lead to a significant overrepresentation of prognostic genes. A striking result is obtained with the outlier-sum statistic (p < 10-12). Many genes with heavy tails generate subgroups of patients with different prognosis. CONCLUSIONS: Genes with high bimodality scores are promising candidates for defining prognostic patient subgroups from expression data. We discuss advantages and disadvantages of the different scores for prognostic purposes. The outlier-sum statistic may be particularly valuable for the identification of genes to be included in prognostic signatures. Among the genes identified as bimodal in the breast cancer data set several have not yet previously been recognized to be prognostic and bimodally expressed in breast cancer.

Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration.

Only little is known about how cells coordinately behave to establish functional tissue structure and restore microarchitecture during regeneration. Research in this field is hampered by a lack of techniques that allow quantification of tissue architecture and its development. To bridge this gap, we have established a procedure based on confocal laser scans, image processing, and three-dimensional tissue reconstruction, as well as quantitative mathematical modeling. As a proof of principle, we reconstructed and modeled liver regeneration in mice after damage by CCl(4), a prototypical inducer of pericentral liver damage. We have chosen the regenerating liver as an example because of the tight link between liver architecture and function: the complex microarchitecture formed by hepatocytes and microvessels, i.e. sinusoids, ensures optimal exchange of metabolites between blood and hepatocytes. Our model captures all hepatocytes and sinusoids of a liver lobule during a 16 days regeneration process. The model unambiguously predicted a so-far unrecognized mechanism as essential for liver regeneration, whereby daughter hepatocytes align along the orientation of the closest sinusoid, a process which we named "hepatocyte-sinusoid alignment" (HSA). The simulated tissue architecture was only in agreement with the experimentally obtained data when HSA was included into the model and, moreover, no other likely mechanism could replace it. In order to experimentally validate the model of prediction of HSA, we analyzed the three-dimensional orientation of daughter hepatocytes in relation to the sinusoids. The results of this analysis clearly confirmed the model prediction. We believe our procedure is widely applicable in the systems biology of tissues.

Dexamethasone-dependent versus -independent markers of epithelial to mesenchymal transition in primary hepatocytes.

Recently, epithelial to mesenchymal transition (EMT) has been shown to represent a feature of dedifferentiating hepatocytes in vitro. Three-dimensional soft collagen gels can antagonize but not completely abolish this effect. Hormonal additives to culture media are known to maintain differentiated hepatocyte functions. Therefore, we studied whether insulin and dexamethasone antagonize EMT in cultured hepatocytes. Both hormones antagonized but not completely abolished certain morphological features of EMT. Dexamethasone antagonized acquisition of fibroblastoid shape, whereas insulin favored bile canaliculi formation. In a subsequent step, we analyzed expression of a battery of EMT-related genes. Of all markers tested, vimentin and snail-1 correlated best with morphological features of EMT. Interestingly, dexamethasone reduced expression levels of both vimentin and snail-1, whereas the influence of insulin was less pronounced. An important result of this study is that 12 out of 17 analyzed EMT markers were transcriptionally influenced by dexamethasone (vimentin, snail-1, snail-2, HNF4 alpha, Twist-1, ZEB2, fibronectin, occludin, MMP14, claudin-1, cytokeratin-8, and cytokeratin-18), whereas the remaining factors seemed to be less dependent on dexamethasone. In conclusion, EMT markers in hepatocytes can be classified as dexamethasone-dependent versus -independent.

ERBB2 induces an antiapoptotic expression pattern of Bcl-2 family members in node-negative breast cancer.

PURPOSE: Members of the Bcl-2 family act as master regulators of mitochondrial homeostasis and apoptosis. We analyzed whether ERBB2 influences the prognosis of breast cancer by influencing the proapoptotic versus antiapoptotic balance of Bcl-2 family members. EXPERIMENTAL DESIGN: ERBB2-regulated Bcl-2 family members were identified by inducible expression of ERBB2 in MCF-7 breast cancer cells and by correlation analysis with ERBB2 expression in breast carcinomas. The prognostic relevance of ERBB2-regulated and all additional Bcl-2 family members was determined in 782 patients with untreated node-negative breast cancer. The biological relevance of ERBB2-induced inhibition of apoptosis was validated in a murine tumor model allowing conditional ERBB2 expression. RESULTS: ERBB2 caused an antiapoptotic phenotype by upregulation of MCL-1, TEGT, BAG1, BNIP1, and BECN1 as well as downregulation of BAX, BMF, BNIPL, CLU, and BCL2L13. Upregulation of the antiapoptotic MCL-1 [P = 0.001, hazard ratio (HR) 1.5] and BNIP3 (P = 0.024; HR, 1.4) was associated with worse prognosis considering metastasis-free interval, whereas clusterin (P = 0.008; HR, 0.88) and the proapoptotic BCL2L13 (P = 0.019; HR, 0.45) were associated with better prognosis. This indicates that ERBB2 alters the expression of Bcl-2 family members in a way that leads to adverse prognosis. Analysis of apoptosis and tumor remission in a murine tumor model confirmed that the prototypic Bcl-2 family member Bcl-x(L) could partially substitute for ERBB2 to antagonize tumor remission. CONCLUSIONS: Our results support the concept that ERBB2 influences the expression of Bcl-2 family members to induce an antiapoptotic phenotype. Antagonization of antiapoptotic Bcl-2 family members might improve breast cancer therapy, whereby MCL-1 and BNIP3 represent promising targets.

Munich Oktoberfest experience: remarkable impact of sex and age in ethanol intoxication.

Approximately 5,000 of 6 million annual visitors of the Oktoberfest in Munich have to undergo medical treatment. Patients with alcohol intoxication without trauma or further complications are all treated in a specialized medical camp. We studied these patients in order to identify risk factors and to assess the relevance of the Glasgow Coma Score (GCS) and of ethanol blood concentrations for patient management. In 2004 totally 405 patients suffering from ethanol intoxication without trauma were treated in the medical camp. A complete set of the following data was obtained from all 405 patients: GCS, ethanol blood concentration, age, sex, blood pressure (mean, systolic and diastolic), body temperature, heart rate, blood sugar, GOT, gamma-GT, and CK. A multivariate logistic regression model was applied to identify risk factors predicting patients at increased risk of hospitalization. Low GCS (< or =8 vs. >8, OR: 4.18, CI: 1.96-8.65) low age (20-29 vs. > or =30 years, OR: 2.35, CI: 1.05-5.65) and male gender (male vs. female, OR: 3.58, CI: 1.36-9.34) independently predicted patients that had to be hospitalized. All other parameters including ethanol blood concentrations were not explanatory. Patients with GCS < or = 8 (n = 66) had a lower median blood pressure (P = 0.0312) and showed a smaller increase in blood pressure during the observation period compared to patients with GCS > 8 (P < 0.001), suggesting that this subgroup may require longer recovery periods. Men aged 20-29 years were at highest risk for hospital admission. Increased risk could not be explained by higher ethanol blood concentrations in this subgroup. Importantly, GCS < 6 does not justify endotracheal intubation in ethanol intoxicated patients, when further complications, such as trauma, can be excluded.

Cadmium, cobalt and lead cause stress response, cell cycle deregulation and increased steroid as well as xenobiotic metabolism in primary normal human bronchial epithelial cells which is coordinated by at least nine transcription factors.

Workers occupationally exposed to cadmium, cobalt and lead have been reported to have increased levels of DNA damage. To analyze whether in vivo relevant concentrations of heavy metals cause systematic alterations in RNA expression patterns, we performed a gene array study using primary normal human bronchial epithelial cells. Cells were incubated with 15 microg/l Cd(II), 25 microg/l Co(II) and 550 microg/l Pb(II) either with individual substances or in combination. Differentially expressed genes were filtered out and used to identify enriched GO categories as well as KEGG pathways and to identify transcription factors whose binding sites are enriched in a given set of promoters. Interestingly, combined exposure to Cd(II), Co(II) and Pb(II) caused a coordinated response of at least seven stress response-related transcription factors, namely Oct-1, HIC1, TGIF, CREB, ATF4, SRF and YY1. A stress response was further corroborated by up regulation of genes involved in glutathione metabolism. A second major response to heavy metal exposure was deregulation of the cell cycle as evidenced by down regulation of the transcription factors ELK-1 and the Ets transcription factor GABP, as well as deregulation of genes involved in purine and pyrimidine metabolism. A third and surprising response was up regulation of genes involved in steroid metabolism, whereby promoter analysis identified up regulation of SRY that is known to play a role in sex determination. A forth response was up regulation of xenobiotic metabolising enzymes, particularly of dihydrodiol dehydrogenases 1 and 2 (AKR1C1, AKR1C2). Incubations with individual heavy metals showed that the response of AKR1C1 and AKR1C2 was predominantly caused by lead. In conclusion, we have shown that in vivo relevant concentrations of Cd(II), Co(II) and Pb(II) cause a complex and coordinated response in normal human bronchial epithelial cells. This study gives an overview of the most responsive genes.

Tracking of human cells in mice.

Tracking and tracing of transplanted cells in mice is required in many fields of research. Examples are transplantation of stem cells into organs of mice to study their differentiation capacity and injection of tumor cells to examine metastatic behavior. In the present study we tested the lipid dye CM-DiI and red fluorescent nanoparticles Qdot655 for their applicability in tagging and tracing of human cells in mice. Labeling of different cell types, including MCF-7 human breast cancer cells, human cord blood derived cells, human NeoHep cells and human hepatopancreatic precursor cells, is technically easy and did not compromise further cell culture. After transplantation of CM-DiI or Qdot655 marked cells, red fluorescent structures could be detected already in unprocessed paraffin slices of the studied organs, namely liver, lung, pancreas, kidney, spleen and bone marrow. Next, we examined whether the red fluorescent structures represent the transplanted human cells. For this purpose, we established an in situ hybridization (ISH) technique that allows clear-cut differentiation between human and murine nuclei, based on simultaneous hybridization with human alu and mouse major satellite (mms) probes. We observed a high degree of coincidence between CM-DiI-marked cells and alu positive nuclei. However, also some mms positive cells contained CM-DiI, suggesting phagocytosis of the transplanted CM-DiI-marked cells. The degree of such CM-DiI-positive mouse cells depended on the cell type and route of administration. From a technical point of view it was important that CM-DiI-positive structures in paraffin slices remained fluorescent also after ISH. In contrast, Qdot655 positive structures faded during further staining procedures. In conclusion, marking of cells with CM-DiI or Qdot655 prior to transplantation facilitates recovery of human cells, since a high fraction of positive structures in the host's tissue originate from the transplanted cells. However, CM-DiI or Qdot655 positive staining of individual cells in transplanted tissues is not sufficient to prove their human origin. Additional procedures, such as ISH with alu-probes, are essential, when characterizing individual cells.

Fate of extrahepatic human stem and precursor cells after transplantation into mouse livers.

UNLABELLED: In recent years, a large number of groups studied the fate of human stem cells in livers of immunodeficient animals. However, the interpretation of the results is quite controversial. We transplanted 4 different types of human extrahepatic precursor cells (derived from cord blood, monocytes, bone marrow, and pancreas) into livers of nonobese diabetic/severe combined immunodeficiency mice. Human hepatocytes were used as positive controls. Tracking of the transplanted human cells could be achieved by in situ hybridization with alu probes. Cells with alu-positive nuclei stained positive for human albumin and glycogen. Both markers were negative before transplantation. However, cells with alu-positive nuclei did not show a hepatocyte-like morphology and did not express cytochrome P450 3A4, and this suggests that these cells represent a mixed cell type possibly resulting from partial transdifferentiation. Using antibodies specific for human albumin, we also observed a second human albumin-positive cell type that could be clearly distinguished from the previously described cells by its hepatocyte-like morphology. Surprisingly, these cells had a mouse and not a human nucleus which is explained by transdifferentiation of human cells. Although it has not yet been formally proven, we suggest horizontal gene transfer as a likely mechanism, especially because we observed small fragments of human nuclei in mouse cells that originated from deteriorating transplanted cells. Qualitatively similar results were obtained with all 4 human precursor cell types through different routes of administration with and without the induction of liver damage. CONCLUSION: We observed evidence not for transdifferentiation but instead for a complex situation including partial differentiation and possibly horizontal gene transfer.

Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers.

AIMS: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo. METHODS: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration. CONCLUSION: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.