BACKGROUND

Mesalazine (mesalamine) (5-ASA) is considered an anti-inflammatory drug for the treatment of inflammatory bowel disease. It is well tolerated by most patients and induces mucosal healing specifically in ulcerative colitis. Besides its anti-inflammatory properties, 5-ASA has been studied for cancer inhibitory activities as it seems to reduce colorectal cancer incidence in patients using this drug for long periods of time. However, detailed molecular mechanisms of drug action are vague.

OBJECTIVE

To evaluate known molecular mechanisms of 5-ASA on chemoprevention of colorectal malignancy.

METHODS

Systematic review with search terms '5 aminosalicylic acid, mesalazine, 5-ASA, mesalazine, molecular mechanisms, chemoprevention' between 2006 and August 2009.

RESULTS

A total of 48 studies were retrieved that link 5-ASA chemopreventive properties to five distinct pathways. These include interference with cell cycle progression (<em>12</em> references), scavenging of reactive oxygen- or nitrogen-derived metabolites (16 references), TNF-alpha/TGF-ss signalling (11 references), <em>WNT</em>/beta-catenin signalling (5 references) and anti-bacterial properties (4 references).

CONCLUSIONS

In the recent years, a large amount of molecular data has accumulated supporting the notion that 5-ASA biological effects interfere with colorectal cancer development. These molecular pathways are of special interest in the search for 5-ASA's molecular target(s) and development of novel chemopreventive compounds.

Calorie restriction (CR) delays aging and onset of age-related diseases in a variety of organisms from yeast to mammals. However, the molecular mechanism underlying the CR effect remains to be elucidated. It is well known that the hypothalamus is an important component of the brain neuroendocrine system for the regulation of the aging process. In this report, we have systematically examined the gene expression profiles of hypothalami from 5-, <em>12</em>-, 19- and 24-month-old mice fed ad libitum or subjected to CR since weaning. Our results demonstrated that CR significantly altered the expression level of 490 genes in an age-dependent manner, with the greatest impact at middle age. Classification based on functional analysis indicated that a large number of these genes were involved in brain development and neurogenesis, including genes involved in Wingless (<em>Wnt</em>) and Notch signaling pathways. In addition, the expression levels of numerous genes involved in the stress and inflammatory responses, as well as apoptosis, were affected by CR. Interestingly, we found that a number of genes involved in the stress response and apoptosis were down-regulated in early but up-regulated in late stage CR. The most notable finding was that CR altered the expression of genes associated with the mammalian target of the rapamycin (mTOR) nutrient sensing pathway, which has recently been shown to be involved in the regulation of energy intake and aging. By applying rapamycin, a specific pharmacological inhibitor of mTOR signaling, we found that the inhibition of mTOR could significantly prevent neuronal apoptosis induced by Paraquat. Taken together, our results provided not only a systematic expression profile of the hypothalamic response to CR, but also revealed the linkage between CR and mTOR signaling in the neuroprotection in mice.

Our previous studies have implicated the <em>Wnt</em> antagonist, sFRP1, as a tumour suppressor gene in advanced colorectal cancer. In this study, we set out to investigate the relationship between sFRP1 expression and large bowel adenomas, a precursor of colorectal cancer. The induction of beta-catenin/TCF mediated transcription is both a frequent early event in colorectal neoplasia, and a key downstream effect of <em>wnt</em> growth factor signalling. Lithium treatment of a small bowel mucosal cell line (FHs 74 int) induced sFRP1 within 8 h, indicating that this gene is positively regulated by beta-catenin, contrasting with the suppression of sFRP1 expression, we saw previously in advanced colorectal cancers. We therefore investigated a series of <em>12</em> adenomas and matched large bowel mucosa samples. Real-time RT-PCR analysis showed a reduction in sFRP1 expression in all <em>12</em> dysplastic lesions (median 485-fold, IQR <em>12</em>0- to 1500-fold), indicating factors other than beta-catenin influence sFRP1 levels. In a second series of 11 adenomas, we identified methylation of the sFRP1 promotor region in all 11 samples, and this was increased compared with the surrounding normal mucosa in seven cases. Immunohistochemical analysis using a polyclonal antibody supported these findings, with sFRP1 expression reduced in many of the adenoma samples examined. sFRP1 staining in normal mucosa adjacent to the dysplastic tissue was also reduced compared with the normal controls, suggesting that sFRP1 expression may be suppressed in a field of mucosa rather than in individual cells. This study identifies sFRP1 inactivation at the premalignant stage of colorectal cancer development, indicating that these pathways may be useful targets for chemoprevention strategies in this common solid tumour.

β-Arrestins (Arrb) participate in the regulation of multiple signaling pathways, including <em>Wnt</em>/β-catenin, the major actor in human colorectal cancer initiation. To better understand the roles of Arrb in intestinal tumorigenesis, a reverse genetic approach (Arrb(-/-)) and in vivo siRNA treatment were used in Apc(Δ14/+) mice. Mice with Arrb2 depletion (knockout and siRNA) developed only 33% of the tumors detected in their Arrb2-WT littermates, whereas Arrb1 depletion remained without significant effect. These remaining tumors grow normally and are essentially Arrb2-independent. Unsupervised hierarchical clustering analysis showed that they clustered with 25% of Apc(Δ14/+);Arrb2(+/+) tumors. Genes overexpressed in this subset reflect a high interaction with the immune system, whereas those overexpressed in Arrb2-dependent tumors are predominantly involved in <em>Wnt</em> signaling, cell adhesion, migration, and extracellular matrix remodeling. The involvement of Arrb2 in intestinal tumor development via the regulation of the <em>Wnt</em> pathway is supported by ex vivo and in vitro experiments using either tumors from Apc(Δ14/+) mice or murine Apc(Min/+) cells. Indeed, Arrb2 siRNAs decreased the expression of <em>Wnt</em> target genes in cells isolated from <em>12</em> of 18 tumors from Apc(Δ14/+) mice. In Apc(Min/+) cells, Arrb2 siRNAs completely reversed the increased <em>Wnt</em> activity and colony formation in soft agar induced by Apc siRNA treatment, whereas they did not affect these parameters in basal conditions or in cells expressing constitutively active β-catenin. We demonstrate that Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated <em>Wnt</em> pathway activity and identify a previously unsuspected molecular heterogeneity among tumors induced by truncating Apc mutations.

Colorectal cancer is one of the leading causes of cancer deaths. It correlates to a high fat diet, which causes an increase of the secondary bile acids including deoxycholate (DOC) in the intestine. We aimed to determine the effects of DOC on intestinal carcinogenesis in Apc (min/+) mice, a model of spontaneous intestinal adenomas. Four-week old Apc (min/+) mice were treated with 0.2 % DOC in drinking water for <em>12</em> weeks. The number and size of tumors were measured, and tissue sections were prepared for the evaluation of intestinal carcinogenesis, cell proliferation, and apoptosis. The activation of <em>Wnt</em> signaling was detected in the intestinal tumor cells of the Apc (min/+) mice, and also in the human colon samples. DOC increased the number of intestine tumors by 165.1 % compared with that in untreated Apc (min/+) mice mainly in the middle and distal segments of the small intestine and colon. The numbers of all sizes of tumors in the small intestine were increased. Intestinal carcinogenesis was confirmed in 75 % mice in DOC treated-Apc (min/+) mice compared with 0 % in untreated mice. This was accompanied by promoting tumor cell proliferation and decreasing apoptosis, and increasing the percentage of β-catenin positive cells and its nuclear expression in intestinal tumor cells of Apc (min/+) mice, and also up-regulating the expression of cyclin D1. In addition, the activation of <em>Wnt</em> signaling also played in modulating human colon carcinogenesis. Our studies suggest that DOC enhances the multiplicity of intestinal tumor, and accelerates intestinal adenoma-adenocarcinoma sequence in Apc (min/+) mice mediated by stimulating tumor cell proliferation and decreasing apoptosis through enhancing <em>Wnt</em> signaling.

Recently, heterozygous mutations in exon 2 of the mediator complex subunit <em>12</em> gene have been described in 50% to 70% of uterine leiomyomas; the recurrent nature of these mutations suggests an important role in their pathogenesis. Mediator complex subunit <em>12</em> is involved in regulation of transcription and <em>Wnt</em> signaling. So far, little is known about the pathogenesis of the different subtypes of extrauterine leiomyomas and leiomyosarcomas. We performed mutation analysis of mediator complex subunit <em>12</em> and immunohistochemistry for β-catenin, using 69 tumors of 64 patients including 19 uterine leiomyomas, 6 abdominal leiomyomas, 9 angioleiomyomas, 5 piloleiomyomas, and 7 uterine and 23 soft tissue leiomyosarcomas. In line with previous observations, 58% of uterine leiomyomas carried a mediator complex subunit <em>12</em> mutation. However, all other extrauterine leiomyomas were negative with the exception of 1 abdominal leiomyoma with a likely primary uterine origin. Of the 30 leiomyosarcomas, only 1 uterine tumor harbored a mutation. A new observation is the identification of 3 tumors with a homozygous mutation; a monosomy X or interstitial deletion was excluded. β-Catenin immunohistochemistry showed nuclear positivity in only 55% of the mediator complex subunit <em>12</em>-mutated uterine leiomyomas, suggesting the involvement of pathways other than canonical <em>Wnt</em> signaling in tumorigenesis. Interestingly, 80% of mediator complex subunit <em>12</em> wild-type sporadic piloleiomyomas displayed nuclear β-catenin positivity, indicating its involvement in this leiomyoma subtype. The lack of mediator complex subunit <em>12</em> mutations in extrauterine leiomyomas and leiomyosarcomas indicates that these tumors arise through a different pathway, emphasizing the genetic heterogeneity of smooth muscle tumors.

GIPC1/RGS19IP1/GIPC, GIPC2, and GIPC3 are a family of central PDZ-domain proteins with GH1 and GH2 domains. GIPC1 interacts with GTPase-activating protein RGS19/RGS-GAIP, TGFbeta type III receptor, receptor tyrosine kinase TrkA, and integrin alpha6A subunit. Xenopus homologue of human GIPCs interacts with Frizzled-3 class of <em>WNT</em> receptor. We investigated expression of human GIPC1 mRNA in normal tissues, cancer cell lines, and primary tumors. GIP1A probe (nucleotide position 1075-1483 of GIPC1 cDNA) hybridized to GIPC1 mRNA of 1.8 kb in size. GIPC1 mRNA was almost ubiquitously expressed in various normal tissues. Expression level of GIPC1 mRNA was relatively lower in bone marrow and peripheral blood leukocytes. GIPC1 mRNA was relatively highly expressed in gastric cancer cell lines OKAJIMA, TMK1, MKN28, MKN45, MKN74, KATO-III, pancreatic cancer cell line AsPC-1, colorectal cancer cell line SW480, and lung cancer cell line A549. On the other hand, GIPC1 mRNA was almost undetectable in leukemia/lymphoma cell lines HL-60, Raji, and Daudi. Expression of GIPC1 mRNA was down-regulated in <em>12</em> out of 14 cases of primary kidney tumors, 10 out of 18 cases of primary colorectal tumors, 3 out of 8 cases of primary gastric cancer, 3 out of 3 cases of primary prostate cancer. Because GIPC1 induces increased expression of TGFbeta type III receptor at the cell surface and enhanced responsiveness to TGFbeta, down-regulation of GIPC1 mRNA in tumors might promote cellular proliferation through interference of TGFbeta signaling.

wingless (wg) and its vertebrate homologues, the <em>Wnt</em> genes, play critical roles in the generation of embryonic pattern. In the developing Drosophila epidermis, wg is expressed in a single row of cells in each segment, but it influences cell identities in all rows of epidermal cells in the 10- to <em>12</em>-cell-wide segment. Wg signaling promotes specification of two distinct aspects of the wild-type intrasegmental pattern: the diversity of denticle types present in the anterior denticle belt and the smooth or naked cuticle constituting the posterior surface of the segment. We have manipulated the expression of wild-type and mutant wg transgenes to explore the mechanism by which a single secreted signaling molecule can promote these distinctly different cell fates. We present evidence consistent with the idea that naked cuticle cell fate is specified by a cellular pathway distinct from the denticle diversity-generating pathway. Since these pathways are differentially activated by mutant Wg ligands, we propose that at least two discrete classes of receptor for Wg may exist, each transducing a different cellular response. We also find that broad Wg protein distribution across many cell diameters is required for the generation of denticle diversity, suggesting that intercellular transport of the Wg protein is an essential feature of pattern formation within the epidermal epithelium. Finally, we demonstrate that an 85 amino acid region not conserved in vertebrate <em>Wnts</em> is dispensable for Wg function and we discuss structural features of the Wingless protein required for its distinct biological activities.

The <em>Wnt</em>/β-catenin pathway has been known to play a role in induction of immune tolerance, but its role in the induction and maintenance of natural killer T (NKT) cell anergy is unknown. We found that activation of the <em>Wnt</em> pathways in the liver microenvironment is important for induction of NKT cell anergy. We identified a number of stimuli triggering <em>Wnt</em>/β-catenin pathway activation, including exogenous NKT cell activator, glycolipid α-GalCer, and endogenous prostaglandin E2 (PGE2). Glycolipid α-GalCer treatment of mice induced the expression of wnt3a and wnt5a in the liver and subsequently resulted in a liver microenvironment that induced NKT cell anergy to α-GalCer restimulation. We also found that circulating PGE2 carried by nanoparticles is stable, and that these nanoparticles are A33(+) . A33(+) is a marker of intestinal epithelial cells, which suggests that the nanoparticles are derived from the intestine. Mice treated with PGE2 associated with intestinal mucus-derived exosome-like nanoparticles (IDENs) induced NKT cell anergy. PGE2 treatment leads to activation of the <em>Wnt</em>/β-catenin pathway by inactivation of glycogen synthase kinase 3β of NKT cells. IDEN-associated PGE2 also induces NKT cell anergy through modification of the ability of dendritic cells to induce interleukin-<em>12</em> and interferon-β in the context of both glycolipid presentation and Toll-like receptor-mediated pathways.

CONCLUSIONS

These findings demonstrate that IDEN-associated PGE2 serves as an endogenous immune modulator between the liver and intestines and maintains liver NKT cell homeostasis. This finding has implications for development of NKT cell-based immunotherapies. (HEPATOLOGY 2013).

Current endeavors in the type 2 diabetes (T2D) field include gaining a better understanding of extracellular signaling pathways that regulate pancreatic islet function. Recent data suggest that both Bmp and <em>Wnt</em> pathways are operative in pancreatic islets and play a positive role in insulin secretion and glucose homeostasis. Our laboratory found the dual Bmp and <em>Wnt</em> antagonist Sostdc1 to be upregulated in a mouse model of islet dysmorphogenesis and nonimmune-mediated lean diabetes. Because Bmp signaling has been proposed to enhance β-cell function, we evaluated the role of Sostdc1 in adult islet function using animals in which Sostdc1 was globally deleted. While Sostdc1-null animals exhibited no pancreas development phenotype, a subset of mutants exhibited enhanced insulin secretion and improved glucose homeostasis compared with control animals after <em>12</em>-wk exposure to high-fat diet. Loss of Sostdc1 in the setting of metabolic stress results in altered expression of Bmp-responsive genes in islets but did not affect expression of <em>Wnt</em> target genes, suggesting that Sostdc1 primarily regulates the Bmp pathway in the murine pancreas. Furthermore, our data indicate that removal of Sostdc1 enhances the downregulation of the closely related Bmp inhibitors Ctgf and Gremlin in islets after 8-wk exposure to high-fat diet. These data imply that Sostdc1 regulates expression of these inhibitors and provide a means by which Sostdc1-null animals show enhanced insulin secretion and glucose homeostasis. Our studies provide insights into Bmp pathway regulation in the endocrine pancreas and reveal new avenues for improving β-cell function under metabolic stress.

Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by interactions between MM cells and the bone marrow microenvironment and is characterized by increased osteoclastic bone resorption and decreased osteoblastic bone formation. Recently, the role of osteoblast inhibition has come into focus, especially the possible role of overexpression of DKK1, an inhibitor of the <em>Wnt</em> signalling pathway. Further, CKS2, PSME2 and DHFR have also been reported as candidate genes for OBD. We studied the gene expression by quantitative reverse transcription polymerase chain reaction of TNFSF11 (RANKL), TNFSF11A (RANK), TNFRSF11B (OPG), CCL3 (MIP1A), CCL4 (MIP1B), PTHR1 (PTHrp), DKK1, CKS2, PSME2 and DHFR in purified, immunophenotypic FACS-sorted plasma cells from 171 newly diagnosed MM patients, 20 patients with monoclonal gammopathy of undetermined significance and <em>12</em> controls. The gene expressions of the analysed genes were correlated with radiographically assessed OBD. Only overexpression of DKK1 was correlated to the degree of OBD. Myeloma cells did not express TNFSF11A, TNFSF11, or TNFRSF11B, and very rarely expressed CCL3 and PTHR11. CCL4, CKS2, PSME2 and DHFR were variably expressed, but the expression of these genes showed no correlation with OBD. In contrast, loss of PSME2 expression in MM plasma cells was significantly correlated with OBD.

BACKGROUND

Animal models suggest that the osteoblast-stimulating actions of PTH are mediated by acute suppression of sclerostin, an inhibitor of the anabolic Wnt pathway. The immediate physiological changes in serum sclerostin in response to PTH infusion have not been reported in human studies.

OBJECTIVE

We sought to determine the acute physiological effects of PTH infusion on serum sclerostin and bone turnover markers in healthy adult men.

METHODS

Fifty-three healthy adult men underwent an 18-h iv infusion of human PTH(1-34) at a dose of 0.55 U/kg · h.

RESULTS

Serum levels of ionized calcium, sclerostin, and markers of bone formation (osteocalcin and amino-terminal propeptide of type I procollagen) and bone resorption (C-telopeptide and N-telopeptide) were obtained at 0, 6, 12, and 18 h.

RESULTS

Serum ionized calcium, C-telopeptide, and N-telopeptide increased, and osteocalcin and amino-terminal propeptide of type I procollagen fell linearly throughout the PTH infusion (P < 0.001 for all). Average ± sem sclerostin levels declined from 936 ± 65 to 813 ± 63 pg/ml at 6 h (P < 0.001) and remained stably suppressed for the duration of the PTH infusion. There were no significant correlations between change in sclerostin and change in bone markers.

CONCLUSIONS

Serum sclerostin declined in response to acute PTH infusion within 6 h in healthy adult men. The early plateau in sclerostin suppression may indicate that maximal stimulation of the Wnt pathway is achieved quickly after exposure to PTH. Our findings support the hypothesis that PTH may mediate its anabolic effects in part via suppression of sclerostin.

The repair of osteochondral defects can be enhanced with scaffolds but is often accompanied with undesirable terminal differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Parathyroid hormone-related protein (PTHrP) has been shown to inhibit aberrant differentiation, but administration at inappropriate time points would have adverse effects on chondrogenesis. This study aims to develop an effective tissue engineering strategy by combining PTHrP and collagen-silk scaffold for osteochondral defect repair. The underlying mechanisms of the synergistic effect of combining PTHrP administration with collagen-silk scaffold implantation for rabbit knee joint osteochondral defect repair were investigated. In vitro studies showed that PTHrP treatment significantly reduced Alizarin Red staining and expression of terminal differentiation-related markers. This is achieved in part through blocking activation of the canonical <em>Wnt</em>/β-catenin signaling pathway. For the in vivo repair study, intra-articular injection of PTHrP was carried out at three different time windows (4-6, 7-9 and 10-<em>12</em> weeks) together with implantation of a bi-layer collagen-silk scaffold. Defects treated with PTHrP at the 4-6 weeks time window exhibited better regeneration (reconstitution of cartilage and subchondral bone) with minimal terminal differentiation (hypertrophy, ossification and matrix degradation), as well as enhanced chondrogenesis (cell shape, Col2 and GAG accumulation) compared with treatment at other time windows. Furthermore, the timing of PTHrP administration also influenced PTHrP receptor expression, thus affecting the treatment outcome. Our results demonstrated that intra-articular injection of PTHrP at 4-6 weeks post-injury together with collagen-silk scaffold implantation is an effective strategy for inhibiting terminal differentiation and enhancing chondrogenesis, thus improving cartilage repair and regeneration in a rabbit model.

The <em>Wnts</em> can be classified into two classes based on their ability to transform cells. The Wnt5a class can antagonize the effects of transforming <em>Wnts</em> partly through effects on cell migration. To understand the mechanisms of regulation of Wnt5a, we investigated its expression in human normal and breast cancer cell lines. Elevation of Wnt5a in HB2, a normal breast epithelial cell line, was linearly correlated with cell density, but this did not occur in cancer cell lines. We examined intracellular events responsible for the regulation of Wnt5a by cell to cell contacts, using various metabolic agents known to affect signal transduction pathways. Agents that selectively blocked protein kinase C (calphostin C) or protein tyrosine kinases (genistein) reduced the level of Wnt5a expression markedly. Protein kinase C activation by phorbol <em>12</em>-myristate 13-acetate up-regulated Wnt5a partly through prolongation of Wnt5a mRNA half-life. Cytoskeleton reorganization following cytochalasin D treatment caused an induction of Wnt5a, which was associated with changes in cell morphology. Calphostin C did not block these effects, showing that protein kinase C is acting upstream of cytoskeletal modulation. However, the cancer cell lines treated with cytochalasin D showed no changes in cell morphology or Wnt5a induction, suggesting disruption of this regulatory pathway in cancer.

BACKGROUND

The <em>Wnt</em>/β-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of <em>12</em> <em>Wnt</em>/β-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples.

METHODS

Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and β-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models.

RESULTS

For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/β-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of β-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with β-catenin and thus demonstrating its epigenetically regulated inhibition effect.

CONCLUSIONS

The results suggest an epigenetic silencing mechanism of the Wnt/β-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

The potential of arsenic to cause neural tube defects (NTD) in the human population remains a topic of controversy. While clearly toxic, the lack of well-defined human epidemiologic studies on this subject has made it difficult to fully understand the effects arsenic may have on the developing human neural tube. In the absence of good clinical data, we have tried to develop a murine model where hypotheses about the reproductive toxicity of arsenate can be tested. For these studies a murine strain (LM/Bc) that has proven to be susceptible to arsenic-induced NTD was use. Because cellular proliferation is vital for normal neural tube closure (NTC) to occur, in the present study we investigated whether an acute arsenate treatment could alter the expression of several cell cycle genes during murine neurulation. Pregnant LM/Bc dams were injected intraperitoneally on gestation day (GD) 7:<em>12</em> (day:hour) and 8:<em>12</em> with 40 mg/kg of arsenate, a treatment that causes exencephaly in 90 to 100% of the exposed fetuses. Neural tubes were then isolated from both control and arsenic treated embryos at GD 9:00, 9:<em>12</em>, 10:00, and 10:<em>12</em>, which encompasses all the stages of neurulation for this murine strain. Using the molecular techniques of in situ transcription and antisense RNA amplification (RT/aRNA) the expression pattern for bc1-2, p53, wee-1, and <em>wnt</em>-1 was analyzed at each of these time points. In the neural tubes isolated from control embryos, the expression of all four genes was significantly altered as neurulation progressed, demonstrating their developmental regulation. Following arsenate treatment, however, there was a significant upregulation in the expression of bc1-2 and p53 at gestational day 9:0, compared to their control values. The heightened expression of both of these genes suggests that arsenic inhibits cell proliferation, rather than inducing apoptosis, which delayed NTC and ultimately led to the neural tube defects observed in exposed embryos.

Epithelial to mesenchymal transition (EMT) is an integral process in the progression of many epithelial tumors. It involves a coordinated series of events, leading to the loss of epithelial features and the acquisition of a mesenchymal phenotype, resulting in invasion and metastasis. The EMT of hepatocellular carcinoma (HCC) cells is thought to be a key event in intrahepatic dissemination and distal metastasis. In this study, we used <em>12</em>-O-tet-radecanoylphorbol-13-acetate (TPA) to dissect the signaling pathways involved in the EMT of HepG2 hepatocarcinoma cells. The spectacular change in phenotype induced by TPA, leading to a pronounced spindle-shaped fibroblast-like cell morphology, required ERK1/2 activation. This ERK1/2-dependent EMT process was characterized by a loss of E-cadherin function, modification of the cytoskeleton, the acquisition of mesenchymal markers and profound changes to extracellular matrix composition and mobility. Snail was essential for E-cadherin repression, but was not sufficient for full commitment of the TPA-triggered EMT. We found that TPA triggered the formation of a complex between Snail and β-catenin that activated the <em>Wnt</em> pathway. This study thus provides the first evidence for the existence of a complex network governed by the ERK1/2 signaling pathway, converging on the coregulation of Snail and the <em>Wnt</em>/β-catenin pathway and responsible for the onset and the progression of EMT in hepatocellular carcinoma cells.

<em>WNT</em> proteins play key roles in carcinogenesis. We have previously cloned and characterized <em>WNT</em>14 and <em>WNT</em>14B/<em>WNT</em>15. <em>WNT</em>14 and <em>WNT</em>3A genes are clustered on human chromosome 1q42, while <em>WNT</em>14B and <em>WNT</em>3 genes are clustered on human chromosome 17q21. Here, we investigated expression of <em>WNT</em>14 and <em>WNT</em>14B mRNAs in human cancer. <em>WNT</em>14 was significantly up-regulated in 1 out of 9 cases of primary breast cancer. <em>WNT</em>14B was not expressed in primary breast, gastric and colorectal cancers. Among 3 human breast cancer cell lines, <em>WNT</em>14 mRNA was expressed in T-47D cells, and weakly expressed in MCF-7 cells. <em>WNT</em>14 mRNA was also detected in 7 out of 7 pancreatic cancer cell lines, <em>12</em> out of <em>12</em> esophageal cancer cell lines, 4 out of 4 cervical cancer cell lines, and 5 out of 7 brain tumor cell lines by using cDNA-PCR. These results indicate that <em>WNT</em>14 rather than <em>WNT</em>14B is preferentially expressed in various types of human cancer, such as breast cancer, gastric cancer, and pancreatic cancer. <em>WNT</em>14 mRNA was up-regulated by interferon gamma (IFNgamma), but not by tumor necrosis factor alpha (TNFalpha), in MKN45 cells derived from gastric cancer, while expression of <em>WNT</em>14B mRNA was not affected by IFNgamma and TNFalpha in MKN45 cells. Although expression of <em>WNT</em>14 mRNA was not affected by beta-estradiol in MCF-7 cells, <em>WNT</em>14B mRNA was transiently up-regulated by beta-estradiol in MCF-7 cells. These results indicate that <em>WNT</em>14 is a target gene of IFNgamma in MKN45 cells, and that <em>WNT</em>14B is a target gene of estrogen in MCF-7 cells.

BACKGROUND

The purpose of this study was to identify genome-wide single nucleotide variants and mutations in African American patients with colorectal cancer (CRC). There is a need of such studies in African Americans, because they display a higher incidence of aggressive CRC tumors.

METHODS

We performed whole exome sequencing (WES) on DNA from <em>12</em> normal/tumor pairs of African American CRC patient tissues. Data analysis was performed using the software package GATK (Genome Analysis Tool Kit). Normative population databases (eg, 1000 Genomes SNP database, dbSNP, and HapMap) were used for comparison. Variants were annotated using analysis of variance and were validated via Sanger sequencing.

RESULTS

We identified somatic mutations in genes that are known targets in CRC such as APC, BRAF, KRAS, and PIK3CA. We detected novel alterations in the Wnt pathway gene, APC, within its exon 15, of which mutations are highly associated with CRC.

CONCLUSIONS

This WES study in African American patients with CRC provides insight into the identification of novel somatic mutations in APC. Our data suggest an association between specific mutations in the Wnt signaling pathway and an increased risk of CRC. The analysis of the pathogenicity of these novel variants may shed light on the aggressive nature of CRC in African Americans.

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The <em>Wnt</em> family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of <em>Wnt</em> members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed <em>Wnt</em>-5a, <em>Wnt</em>-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-<em>12</em>), and maturation and hypertrophy (from day <em>12</em> on) program of cartilage development. <em>Wnt</em>-7a misexpression severely inhibited chondrogenesis from day 7 onward. <em>Wnt</em>-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all <em>Wnt</em> members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for <em>Wnt</em> signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.

BACKGROUND

Dermal papilla (DP) is the site of expression of various hair growth related genes. Various researches have demonstrated the underlying importance of Wnt proteins and wound growth factors in stimulating DP associated stem cells. Microneedling works by stimulation of stem cells and inducing activation of growth factors.

METHODS

Hundred cases of mild to moderate (III vertex or IV) androgenetic alopecia (AGA) were recruited into 2 groups. After randomization one group was offered weekly microneedling treatment with twice daily 5% minoxidil lotion (Microneedling group); other group was given only 5% minoxidil lotion. After baseline global photographs, the scalp were shaved off to ensure equal length of hair shaft in all. Hair count was done in 1 cm(2) targeted fixed area (marked with tattoo) at baseline and at end of therapy (week 12). The 3 primary efficacy parameters assessed were: Change from baseline hair count at 12 weeks, patient assessment of hair growth at 12 weeks, and investigator assessment of hair growth at 12 weeks. A blinded investigators evaluated global photographic response. The response was assessed by 7- point scale.

RESULTS

(1) Hair counts - The mean change in hair count at week 12 was significantly greater for the Microneedling group compared to the Minoxidil group (91.4 vs 22.2 respectively). (2) Investigator evaluation - Forty patients in Microneedling group had +2 to +3 response on 7-point visual analogue scale, while none showed the same response in the Minoxidil group. (3) Patient evaluation - In the Microneedling group, 41 (82%) patients reported more than 50% improvement versus only 2 (4.5%) patients in the Minoxidil group. Unsatisfied patients to conventional therapy for AGA got good response with Microneedling treatment.

CONCLUSIONS

Dermaroller along with Minoxidil treated group was statistically superior to Minoxidil treated group in promoting hair growth in men with AGA for all 3 primary efficacy measures of hair growth. Microneedling is a safe and a promising tool in hair stimulation and also is useful to treat hair loss refractory to Minoxidil therapy.

<em>WNT</em> signaling pathways play important roles in the behavior of human bone marrow stromal cells. Although <em>WNT</em> expression has been examined in human bone marrow stromal cells (hMSCs) with limited numbers of subjects or from commercial sources, there are conflicting results on <em>WNT</em> gene expression in hMSCs. Furthermore, the effects of age and gender on <em>WNT</em> expression in hMSCs are largely unknown. In this study, we evaluated RNA expression of all the <em>WNT</em> genes in hMSCs from 19 subjects, <em>12</em> women and 7 men, aged from 36 to 85 years. Analysis of <em>WNT</em> gene expression in young and old groups indicated that <em>WNT</em>7B and 14 were expressed significantly higher in the young group. <em>WNT</em>2 and <em>WNT</em>13 showed a trend of higher expression in young group. <em>WNT</em>7B, 13, and 14 were inversely correlated with age. Further analysis for gender-specific difference indicated that <em>WNT</em>16 was expressed significantly higher in men than in women. <em>WNT</em>11 showed a trend of higher expression in hMSCs from women. For the hMSCs from women, <em>WNT</em>13 was inversely correlated with age and <em>WNT</em>4 was positively correlated with age. For the hMSCs from men, <em>WNT</em>7B and <em>WNT</em>14 were inversely correlated with age. These data indicated that most of the age-related <em>WNT</em> genes belong to the canonical <em>WNT</em> signaling pathway. Further, there are gender-specific differences in the expression of <em>WNT</em>4, 7B, 13, 14, and 16 in hMSCs. Age and gender account for many of the sample-to-sample variations in <em>WNT</em> gene expression in human marrow stromal cells.

The in vitro and in vivo effects of several <em>Wnt</em> family members have been studied using stably transfected HC11 cells, a clonal mammary epithelial cell line derived from a midpregnant mouse mammary gland capable of hormone-dependent differentiation in vitro. Differential effects of <em>Wnt</em>-1, <em>Wnt</em>-2, and <em>Wnt</em>-7B expression were observed both on the morphology of confluent HC11 cells and on the pattern of E-cadherin expression. <em>Wnt</em>-7B had no apparent effect on HC11 cell morphology or E-cadherin expression, as compared to mock-transfected HC11 cells. Injection of stably transfected pools of <em>Wnt</em>-1, <em>Wnt</em>-2, <em>Wnt</em>-7B, and mock-transfected cells into the cleared fat pad of syngeneic BALB/c mice generated reproducible outgrowths after 8 or <em>12</em> weeks. Mock-transfected cells produced outgrowths that exhibited some morphologically normal ductal and alveolar-like structures. However, no morphologically normal structures were observed in the fat pads containing <em>Wnt</em>-transfected cells. Instead, these outgrowths were characterized by significant fibrosis, epithelial hyperplasia, and multiple sites of growth. In contrast to the lack of an observed effect in vitro, palpable adenocarcinomas were observed <em>12</em> weeks after injection of the <em>Wnt</em>-7B-transfected HC11 cells. These tumors contained significant regions of hyperplastic and transformed epithelium and lacked the fibrotic phenotype observed in the <em>Wnt</em>-1 and -2 outgrowths. These results support the hypothesis that different <em>Wnt</em> family members may elicit distinct functional effects and reinforce the need to perform simultaneous comparisons of <em>Wnt</em> function both in vitro and in vivo. Stably transfected HC11 cells provide a useful model system in which to elucidate the function of different <em>Wnt</em> family members.

The <em>Wnt</em> pathway is involved in cellular processes linked to either proliferation or differentiation. Therefore small molecules offer an attractive opportunity to modulate this pathway, whereas the key enzyme GSK-3beta is of special interest. In this study, non-symmetrically substituted indolylmaleimides have been synthesized and their ability to function as GSK-3beta inhibitors has been investigated in a human neural progenitor cell line. Among the newly synthesized compounds, the substance IM-<em>12</em> showed a significant activity in several biological tests which was comparable or even outplayed the effects of the known GSK-3beta inhibitor SB-216763. Furthermore the treatment of human neural progenitor cells with IM-<em>12</em> resulted in an increase of neuronal cells. Therefore we conclude that indolylmaleimides act via the canonical <em>Wnt</em> signalling pathway by inhibition of the key enzyme GSK-3beta.