A novel pancreatic tumour and stellate cell 3D co-culture spheroid model.

BACKGROUND: Pancreatic ductal adenocarcinoma is a devastating disease with poor outcome, generally characterized by an excessive stroma component. The purpose of this study was to develop a simple and reproducible in vitro 3D-assay employing the main constituents of pancreatic ductal adenocarcinoma, namely pancreatic stellate and cancer cells. METHOD: A spheroid assay, directly co-culturing human pancreatic stellate cells with human pancreatic tumour cells in 3D was established and characterized by electron microscopy, immunohistochemistry and real-time RT-PCR. In order to facilitate the cell type-specific crosstalk analysis by real-time RT-PCR, we developed a novel in vitro 3D co-culture model, where the participating cell types were from different species, human and mouse, respectively. Using species-specific PCR primers, we were able to investigate the crosstalk between stromal and cancer cells without previous cell separation and sorting. RESULTS: We found clear evidence for mutual influence, such as increased proliferation and a shift towards a more mesenchymal phenotype in cancer cells and an activation of pancreatic stellate cells towards the myofibroblast phenotype. Using a heterospecies approach, which we coined virtual sorting, confirmed the findings we made initially in the human-human spheroids. CONCLUSIONS: We developed and characterized different easy to set up 3D models to investigate the crosstalk between cancer and stroma cells for pancreatic cancer.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.

OBJECTIVE: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. DESIGN: Gemcitabine metabolites were analysed in LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. RESULTS: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. CONCLUSIONS: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine.

Expression and function of the gene encoding the voltage-dependent calcium channel beta3-subunit in the mouse placenta.

Voltage-dependent Ca(2+) channels (VDCC) exist in most excitable cells and their properly regulated activity is essential for critical biological processes as many of these are sensitive to cellular Ca(2+) ion concentration. The ancillary cytoplasmic Ca(2+) channel beta subunits (CACNB) modulate Ca(2+) channel function and are required to enhance the number of functional channels in the plasma membrane. There are four genes encoding CACNB subunits and the gene encoding CACNB3 is over expressed in hyperplastic placentas of mouse interspecies hybrids. To determine the role of CACNB3 in the mouse placenta, we performed an expression and function analysis. Our results show that Cacnb3 exhibits specific spatial and temporal expression in the mouse placenta. Deletion of Cacnb3 does not produce a strong placental phenotype, which may be due to expression of other CACNB subunit encoding genes; however, sporadic occurrence of a labyrinthine architecture phenotype, characterized by reduced density of fetal blood vessels and decrease in pericyte number, could be observed. Down-regulation of Cacnb3 expression did not rescue placental hyperplasia in a model of interspecies hybrid placentas, which indicates that up-regulation in the hyperplastic placentas is a downstream event.

Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing factor Sp1.

A number of transcription factors contain so-called zinc finger domains for the interaction with their cognate DNA sequence. It has been shown that removal of the zinc ions complexed in these zinc fingers abrogates DNA binding and transcription activation. Therefore we wanted to test the hypothesis that the activity of transcription factors could be regulated by physiological chelators of zinc. A prominent candidate for such a chelator is the Cys-rich protein thionein (apometallothionein) that is inducible by heavy metal loads, and by other environmental stimuli. Here we show with DNA binding and in vitro transcription assays that thionein indeed can inactivate the zinc finger-containing Sp1 in a reversible manner. By contrast, transcription factor Oct-1, which binds DNA via a homeo-domain, i.e. a helix-turn-helix motif not involving zinc ions, is refractory to thionein action. We propose that modulation of intracellular thionein concentration is used for the coordinated regulation of a large subset of genes whose transcription depends on zinc finger proteins.

[Use of rapid microscopic sputum diagnosis in clinical practice]. / Zur Anwendung der mikroskopischen Sputumschnelldiagnostik in der klinischen Praxis.

A total of 222 unselected expectorated sputum samples obtained from patients with various diseases of the respiratory tract was examined microscopically following the method of Murray and Washington. Based on standardized criteria of evaluation of gram-stained smears direct microscopic examination is a simple and reliable method providing primary information, and can be recommended for bedside diagnostics.

Two closely spaced promoters are equally activated by a remote enhancer: evidence against a scanning model for enhancer action.

To explain the activation of transcription by a remote enhancer, two models are most often considered, namely looping and scanning. A scanning model, also referred to as 'polymerase entry site' model predicts that for two adjacent promoters the one proximal to an enhancer would be preferentially activated. Preferential activation of the proximal promoter in a tandem promoter arrangement has been found before in several laboratories, including our own, but for technical reasons the data were inconclusive with regards to the enhancer mechanism. In the work presented here, we readdress the question of preferential promoter activation by an enhancer using a more clearly defined system. Two identical promoters were kept closeby in a divergent, or directly repeated orientation. The SV40 enhancer was placed at a great distance on one or the other side of the two promoters, to see whether the enhancer position influenced the relative efficiency of the two promoters in transfected cells. Our finding that the promoter usage is virtually unaffected by the enhancer position does not favor a scanning model, but is compatible with a looping model of enhancer action.

Increased bactericidal macrophage activity induced by immunological stimuli is dependent on interferon (IFN)-gamma. Interference of anti-IFN-gamma but not anti-IFN-alpha/beta with modulation of macrophage activity caused by lymphocytic choriomeningitis virus infection or systemic graft-vs.-host reactions.

During generalized immune responses such as the acute phase of graft-vs.-host reaction (GvHR) or many systemic viral infections the macrophage-monocyte system of mice is activated as demonstrated by their increased bactericidal capacity against Listeria monocytogenes. To study the effect of interferon (IFN)-gamma in maintainance of macrophage activity, lymphocytic choriomeningitis virus (LCMV) primed C57BL/6 mice or (C57BL/10 x B10.BR) F1 mice undergoing a GvHR were treated with polyclonal specific sheep anti-IFN-gamma antiserum. In both cases injection of anti-IFN-gamma resulted in inhibition of the stimulatory effects on macrophages as detected by reduced bactericidal activity when compared to mice treated with normal serum. Treatment with a polyclonal sheep anti-IFN-alpha,beta antiserum on the other hand did not interfere with the activation status of the macrophages. The findings suggest that IFN-gamma is produced both early in a GvHR and during the acute phase of an systemic infection with LCMV, and that it is involved in in vivo modulation of the increased activity of mononuclear phagocytes in immunologically stimulated mice.

The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression.

We have described and cloned previously a factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF-1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment.

Regulation of metallothionein gene expression in Cd- or Zn-adapted RK-13 cells.

We explored the molecular genetics underlying the massive induction of isoMTs by Zn2+ or Cd2+ in metal tolerant rabbit kidney (RK-13) sub-line cells, using band shift assays and Southern blotting analysis. In sub-line cells accommodated to intermediate metal concentrations (100 microM Zn2+; 1-20 microM Cd2+) evidence suggested that the increase in the capacity for isoMT synthesis is brought about by an increased binding activity of the nuclear transcription factors MTF-1 and Sp1. Using quantitative band shift analysis with a mouse MRE-d oligonucleotide probe, the binding of both transcription factors was found to be enhanced two to three times over the binding activity measured in the unexposed parental RK-13 cells. Their increase in binding activity is probably the cause of the overexpression of MT genes and the development of metal tolerance in these cells. In cells tolerant to the highest concentrations of metal the analysis of Southern blot signals revealed MT gene amplification to be the most probable cause of the increased MT production. Thus, in cells of sub-lines growing in the presence of 350 microM Zn2+, two of the isoMT genes were coordinately triplicated and in cells tolerant to 150 microM Cd2+ one isoMT gene was amplified two-fold.

Efficient TGF-beta induction of the Smad7 gene requires cooperation between AP-1, Sp1, and Smad proteins on the mouse Smad7 promoter.

Sma- and Mad-related protein 7 (Smad7) is an antagonist of transforming growth factor-beta (TGF-beta) signaling, which has been shown to be induced by TGF-beta itself and also by other stimuli. In an effort to understand the molecular mechanisms underlying the transcriptional regulation of the Smad7 gene by TGF-beta, we cloned and functionally characterized a mouse genomic DNA fragment encompassing the mouse Smad7 proximal promoter. This region was found to contain a CpG island and to be devoid of a classical TATA box. Cloned upstream of a promoter-lacking luciferase reporter gene, this region conferred robust TGF-beta-induced transcription. Point mutations in a palindromic Smad binding element, abolished TGF-beta inducibility completely. Through the use of electrophoretic mobility shift assays, we showed the presence of Smad2, Smad3, and Smad4 in complexes binding to the Smad binding element. Interestingly, we also found that point mutation and/or deletion of binding sites for the transcription factors activator protein-1 and Sp1 led to an attenuation of the basal promoter activity, as well as of the TGF-beta-mediated induction of Smad7. Taken together, our data imply that Smads, together with activator protein-1 and Sp1 transcription factors, are essential for efficient Smad7 promoter activity.

Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter.

Metallothioneins (MTs) are small cysteine-rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal-responsive element, MRE) present in the promoter region of MT genes. We and others have demonstrated the presence of an MRE-binding factor in HeLa cell nuclear extracts. We found that this factor, termed MTF-1 (MRE-binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. Here we report that the amounts of MTF-1-DNA complexes are elevated several-fold in zinc-treated cells, as measured by bandshift assay. We have also cloned the cDNA of mouse MTF-1, a 72.5 kDa protein. MTF-1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF-1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT-I promoter, even at normal zinc levels.

Cloning, chromosomal mapping and characterization of the human metal-regulatory transcription factor MTF-1.

Metallothioneins (MTs) are small cysteine-rich proteins that bind heavy metal ions such as zinc, cadmium and copper with high affinity, and have been functionally implicated in heavy metal detoxification and radical scavenging. Transcription of metallothioneins genes is induced by exposure of cells to heavy metals. This induction is mediated by metal-responsive promoter elements (MREs). We have previously cloned the cDNA of an MRE-binding transcription factor (MTF-1) from the mouse. Here we present the human cDNA equivalent of this metal-regulatory factor. Human MTF-1 is a protein of 753 amino acids with 93% amino acid sequence identity to mouse MTF-1 and has an extension of 78 amino acids at the C-terminus without counterpart in the mouse. The factors of both species have the same overall structure including six zinc fingers in the DNA binding domain. We have physically mapped the human MTF-1 gene to human chromosome 1 where it localizes to the short arm in the region 1p32-34, most likely 1p33. Both human and mouse MTF-1 when produced in transfected mammalian cells strongly bind to a consensus MRE of metallothionein promoters. However, human MTF-1 is more effective than the mouse MTF-1 clone in mediating zinc-induced transcription.

Retention of PDGFR-beta function in mice in the absence of phosphatidylinositol 3'-kinase and phospholipase Cgamma signaling pathways.

Signal transduction by the platelet-derived growth-factor receptor beta (PDGFR-beta) tyrosine kinase is required for proper formation of vascular smooth muscle cells (VSMC). However, the importance of individual PDGFR-beta signal transduction pathways in vivo is not known. To investigate the role of two of the pathways believed to be critical for PDGF signal transduction, we have generated mice that bear a PDGFR-beta that can no longer activate PI3kinase or PLCgamma. Although these mutant mice have normal vasculature, we provide multiple lines of evidence in vivo and from cells derived from the mutant mice that suggest that the mutant PDGFR-beta operates at suboptimal levels. Our observations indicate that although loss of these pathways can lead to attenuated PDGF-dependent cellular function, certain PDGFR-beta-induced signal cascades are not essential for survival in mice.

Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.

We have shown previously that the heavy metal-responsive transcriptional activator MTF-1 regulates the basal and heavy metal-induced expression of metallothioneins. To investigate the physiological function of MTF-1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF-1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF-1(-/-) embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy-chain subunit of the gamma-glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF-1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF-1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF-1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c-Jun and NF-kappaB (p65/RelA).

Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3' kinase signaling.

Platelet-derived growth factor (PDGF) isoforms lead to mitogenic, survival, and chemotactic responses in a variety of mesenchymal cell types during development and in the adult. We have studied the importance of phosphatidylinositol-3' kinase (PI3K) signaling in these responses by mutating the PI3K-binding sites in the PDGF-beta receptor by gene targeting in embryonic stem cells. Homozygous mutant mice developed normally; however, cells derived from the mutants were less chemotactic and had largely lost their ability to contract collagen gels in response to PDGF. Injection of a mast cell degranulating agent in mice led to a decrease in interstitial fluid pressure resulting in edema formation. In contrast to wild-type mice, mutant mice were unable to normalize the pressure after treatment with PDGF. Taken together, these observations suggest a function for PDGF signaling through PI3K in interstitial fluid homeostasis by modulating the tension between cells and extracellular matrix structures.

Identification of Smad7, a TGFbeta-inducible antagonist of TGF-beta signalling.

TGF-beta signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins. The activated TGF-beta receptor induces phosphorylation of two such proteins, Smad2 and Smad3, which form hetero-oligomeric complex(es) with Smad4/DPC4 that translocate to the nucleus, where they then regulate transcriptional responses. However, the mechanisms by which the intracellular signals of TGF-beta are switched off are unclear. Here we report the identification of Smad7, which is related to Smad6. Transfection of Smad7 blocks responses mediated by TGF-beta in mammalian cells, and injection of Smad7 RNA into Xenopus embryos blocks activin/TGF-beta signalling. Smad7 associates stably with the TGF-beta receptor complex, but is not phosphorylated upon TGF-beta stimulation. TGFbeta-mediated phosphorylation of Smad2 and Smad3 is inhibited by Smad7, indicating that the antagonistic effect of Smad7 is exerted at this important regulatory step. TGF-beta rapidly induces expression of Smad7 mRNA, suggesting that Smad7 may participate in a negative feedback loop to control TGF-beta responses.