Differentiation Potential of Pancreatic Fibroblastoid Cells/Stellate Cells: Effects of Peroxisome Proliferator-Activated Receptor Gamma Ligands

Pancreatic stellate cells have been investigated mostly for their activation process, supposed to support the development of pancreatic disease. Few studies have been presented on reversal of the activation process in vitro. Thiazolidinediones (TZDs) have been used as antidiabetics and have now been reported to exert antifibrotic activity. We tested effects of natural and synthetic ligands of peroxisome proliferator-activated receptor gamma (PPARγ) on human pancreatic fibroblastoid cells (hPFCs) in search for specificity of action. Ciglitazone, as a prototype of TZDs, was shown to have reversible growth inhibitory effects on human pancreatic fibroblastoid cells/stellate cells. Cells treated with ciglitazone for three days showed enhanced lipid content and induction of proteins involved in lipid metabolism. Collagen synthesis was reduced in hPFC. Interaction of PPARγ with DNA binding sites upon ligand binding was shown by gel shift analysis. These findings point toward a potential for adipocyte differentiation in human pancreatic fibroblastoid cells

K-ras Mutation Targeted to Gastric Tissue Progenitor Cells Results in Chronic Inflammation, an Altered Microenvironment, and Progression to Intraepithelial

Chronic infectious diseases, such as Helicobacter pylori infection, can promote cancer in a large part through induction of chronic inflammation. Oncogenic K-ras mutation in epithelial cells activates inflammatory pathways, which could compensate for a lack of infectious stimulus. Gastric histopathology and putative progenitor markers [doublecortin and calcium/calmodulin-dependent protein kinase-like 1 (Dcamkl1) and keratin 19 (K19)] in K19-K-ras-V12 (K19-kras) transgenic mice were assessed at 3, 6, 12, and 18 months of age, in comparison with Helicobacter felis–infected wild-type littermates. Inflammation was evaluated by reverse transcription–PCR of proinflammatory cytokines, and K19-kras mice were transplanted with green fluorescent protein (GFP)–labeled bone marrow. Both H. felis infection and K-ras mutation induced upregulation of proinflammatory cytokines, expansion of Dcamkl1+ cells, and progression to oxyntic atrophy, metaplasia, hyperplasia, and high-grade dysplasia. K19-kras transgenic mice uniquely displayed mucous metaplasia as early as 3 months and progressed to high-grade dysplasia and invasive intramucosal carcinoma by 20 months. In bone marrow–transplanted K19-kras mice that progressed to dysplasia, a large proportion of stromal cells were GFP+ and bone marrow–derived, but only rare GFP+ epithelial cells were observed. GFP+ bone marrow–derived cells included leukocytes and CD45− stromal cells that expressed vimentin or α smooth muscle actin and were often found surrounding clusters of Dcamkl1+ cells at the base of gastric glands. In conclusion, the expression of mutant K-ras in K19+ gastric epithelial cells can induce chronic inflammation and promote the development of dysplasia.National Institutes of Health (U.S.) (Grant NIH 5R01 CA120979-02)National Institutes of Health (U.S.) (Grant R01 DK060694)National Institutes of Health (U.S.) (Grant U01 CA143056)National Institutes of Health (U.S.) (Grant P30 DK050306)Uehara Memorial Foundatio

Neutrophils in cancer: neutral no more

Neutrophils are indispensable antagonists of microbial infection and facilitators of wound healing. In the cancer setting, a newfound appreciation for neutrophils has come into view. The traditionally held belief that neutrophils are inert bystanders is being challenged by the recent literature. Emerging evidence indicates that tumours manipulate neutrophils, sometimes early in their differentiation process, to create diverse phenotypic and functional polarization states able to alter tumour behaviour. In this Review, we discuss the involvement of neutrophils in cancer initiation and progression, and their potential as clinical biomarkers and therapeutic targets

CD34+cells augment endothelial cell differentiation of CD14+endothelial progenitor cells in vitro

Neovascularization by endothelial progenitor cells (EPC) for the treatment of ischaemic diseases has been a topic of intense research. The CD34+ cell is often designated as EPC, because it contributes to repair of ischaemic injuries through neovascularization. However, incorporation of CD34+ cells into the neovasculature is limited, suggesting another role which could be paracrine. CD14+ cells can also differentiate into endothelial cells and contribute to neovascularization. However, the low proliferative capacity of CD14+ cell-derived endothelial cells hampers their use as therapeutic cells. We made the assumption that an interaction between CD34+ and CD14+ cells augments endothelial differentiation of the CD14+ cells. In vitro, the influence of CD34+ cells on the endothelial differentiation capacity of CD14+ cells was investigated. Endothelial differentiation was analysed by expression of endothelial cell markers CD31, CD144, von Willebrand Factor and endothelial Nitric Oxide Synthase. Furthermore, we assessed proliferative capacity and endothelial cell function of the cells in culture. In monocultures, 63% of the CD14+-derived cells adopted an endothelial cell phenotype, whereas in CD34+/CD14+ co-cultures 95% of the cells showed endothelial cell differentiation. Proliferation increased up to 12% in the CD34+/CD14+ co-cultures compared to both monocultures. CD34-conditioned medium also increased endothelial differentiation of CD14+ cells. This effect was abrogated by hepatocyte growth factor neutralizing antibodies, but not by interleukin-8 and monocyte chemoattractant protein-1 neutralizing antibodies. We show that co-culturing of CD34+ and CD14+ cells results in a proliferating population of functional endothelial cells, which may be suitable for treatment of ischaemic diseases such as myocardial infarction

Molecular subtypes of breast cancer are associated with characteristic DNA methylation patterns

Introduction: Five different molecular subtypes of breast cancer have been identified through gene expression profiling. Each subtype has a characteristic expression pattern suggested to partly depend on cellular origin. We aimed to investigate whether the molecular subtypes also display distinct methylation profiles. Methods: We analysed methylation status of 807 cancer-related genes in 189 fresh frozen primary breast tumours and four normal breast tissue samples using an array-based methylation assay. Results: Unsupervised analysis revealed three groups of breast cancer with characteristic methylation patterns. The three groups were associated with the luminal A, luminal B and basal-like molecular subtypes of breast cancer, respectively, whereas cancers of the HER2-enriched and normal-like subtypes were distributed among the three groups. The methylation frequencies were significantly different between subtypes, with luminal B and basal-like tumours being most and least frequently methylated, respectively. Moreover, targets of the polycomb repressor complex in breast cancer and embryonic stem cells were more methylated in luminal B tumours than in other tumours. BRCA2-mutated tumours had a particularly high degree of methylation. Finally, by utilizing gene expression data, we observed that a large fraction of genes reported as having subtype-specific expression patterns might be regulated through methylation. Conclusions: We have found that breast cancers of the basal-like, luminal A and luminal B molecular subtypes harbour specific methylation profiles. Our results suggest that methylation may play an important role in the development of breast cancers

A computed tomography based study on rotational alignment accuracy of the femoral component in total knee arthroplasty using computer-assisted orthopaedic surgery

Rotation of the femoral component in total knee arthroplasty (TKA) is of high importance in respect of the balancing of the knee and the patellofemoral joint. Though it is shown that computer assisted surgery (CAOS) improves the anteroposterior (AP) alignment in TKA, it is still unknown whether navigation helps in finding the accurate rotation or even improving rotation. Therefore the aim of our study was to evaluate the postoperative femoral component rotation on computed tomography (CT) with the intraoperative data of the navigation system. In 20 navigated TKAs the difference between the intraoperative stored rotation data of the femoral component and the postoperative rotation on CT was measured using the condylar twist angle (CTA). This is the angle between the epicondylar axis and the posterior condylar axis. Statistical analysis consisted of the intraclass correlation coefficient (ICC) and Bland-Altman plot. The mean intraoperative rotation CTA based on CAOS was 3.5° (range 2.4–8.6°). The postoperative CT scan showed a mean CTA of 4.0° (1.7–7.2). The ICC between the two observers was 0.81, and within observers this was 0.84 and 0.82, respectively. However, the ICC of the CAOS CTA versus the postoperative CT CTA was only 0.38. Though CAOS is being used for optimising the position of a TKA, this study shows that the (virtual) individual rotational position of the femoral component using a CAOS system is significantly different from the position on a postoperative CT scan

Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes

PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state

Induction of interleukin-8 preserves the angiogenic response in HIF-1 alpha-deficient colon cancer cells

authorHypoxia inducible factor-1 (HIF-1) is considered a crucial mediator of the cellular response to hypoxia through its regulation of genes that control angiogenesis^1, ^2, ^3, ^4. It represents an attractive therapeutic target^5, ^6 in colon cancer, one of the few tumor types that shows a clinical response to antiangiogenic therapy^7. But it is unclear whether inhibition of HIF-1 alone is sufficient to block tumor angiogenesis^8, ^9. In HIF-1_α knockdown DLD-1 colon cancer cells (DLD-1^HIF-kd), the hypoxic induction of vascular endothelial growth factor (VEGF) was only partially blocked. Xenografts remained highly vascularized with microvessel densities identical to DLD-1 tumors that had wild-type HIF-1_α (DLD-1^HIF-wt). In addition to the preserved expression of VEGF, the proangiogenic cytokine interleukin (IL)-8 was induced by hypoxia in DLD-1^HIF-kd but not DLD-1^HIF-wt cells. This induction was mediated by the production of hydrogen peroxide and subsequent activation of NF-_KB. Furthermore, the KRAS oncogene, which is commonly mutated in colon cancer, enhanced the hypoxic induction of IL-8. A neutralizing antibody to IL-8 substantially inhibited angiogenesis and tumor growth in DLD-1^HIF-kd but not DLD-1^HIF-wt xenografts, verifying the functional significance of this IL-8 response. Thus, compensatory pathways can be activated to preserve the tumor angiogenic response, and strategies that inhibit HIF-1α may be most effective when IL-8 is simultaneously targeted

Epidermal Stem Cells Are Defined by Global Histone Modifications that Are Altered by Myc-Induced Differentiation

Activation of Myc induces epidermal stem cells to exit their niche and differentiate into sebocytes and interfollicular epidermis, a process that is associated with widespread changes in gene transcription. We have identified chromatin modifications that are characteristic of epidermal stem cells and investigated the effects of Myc activation. Quiescent stem cells in the interfollicular epidermis and the hair follicle bulge had high levels of tri-methylated histone H3 at lysine 9 and H4 at lysine 20. Chromatin in both stem cell populations was hypoacteylated at histone H4 and lacked mono-methylation of histone H4 at lysine 20. Myc-induced exit from the stem cell niche correlated with increased acetylation at histone H4 and transiently increased mono-methylation at lysine 20. The latter was replaced by epigenetic modifications that are largely associated with chromatin silencing: di-methylation at histone H3 lysine 9 and histone H4 lysine 20. These modifications correlated with changes in the specific histone methyltransferases Set8 and Ash-1. The Myc-induced switch from mono- to di-methylated H4K20 required HDAC activity and was blocked by the HDAC inhibitor trichostatin A (TSA). TSA treatment induced a similar epidermal phenotype to activation of Myc, and activation of Myc in the presence of TSA resulted in massive stimulation of terminal differentiation. We conclude that Myc-induced chromatin modifications play a major role in Myc-induced exit from the stem cell compartment