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Invasiveness of tumor cells is often determined by the profile of their expressed genes. To determine the gene expression differences between an invasive and a non-invasive human breast tumor cells, we selected BT-549 (invasive) and MDA-MB-468 (non-invasive) cells, and compared their transcriptomes by cDNA microarray analysis. Among the significant differences in gene expressions, notable are the up-regulation of cytokeratins 8 and 19, and down-regulation of metallothioneins 1G and IL in MDA-MB-468 cells. Since MDA-MB-468 cells do not express SLUG, a member of a small family of E2-box-binding zinc finger silencer proteins, we studied whether the cytokeratin gene overexpressions in these cells are due to the absence of SLUG. Inducible expression of SLUG in MDA-MB-468 cells inhibited the expressions of the cytokeratin 8 and 19 but not others as was revealed by microarray analysis. Similarly, siRNA knock down of SLUG in BT-549 cells increased the expressions of those cytokeratin mRNAs. SLUG levels in the cell regulated the function of cytokeratins 8 and 19 gene promoters. We conclude that the expressions of cytokeratins and metallothioneins may be associated with the differential invasive behaviors of these breast tumor cells and SLUG may have regulatory roles in this process.
This study characterizes the morphologic features and the endogenous fluorescence in the stratified squamous epithelia of the 7,12-dimethylbenz(a)anthracene-treated hamster cheek pouch model of carcinogenesis using multiphoton laser scanning microscopy (MPLSM). MPLSM allows high-resolution, three-dimensional image data to be collected deeper within thick tissue samples with reduced phototoxicity compared with single-photon imaging. Three-dimensional image stacks of normal (n = 13), precancerous (dysplasia, n = 12; carcinoma in situ, n = 9) and cancerous tissue [nonpapillary squamous cell carcinoma (SCC), n = 10, and papillary SCC, n = 7] sites in the hamster cheek pouch were collected in viable, unsectioned tissue biopsies at a two-photon excitation wavelength of 780 nm. Five features were quantified from the MPLSM images. These included nuclear density versus depth, keratin layer thickness, epithelial thickness, and the fluorescence per voxel in the keratin and epithelial layers. Statistically significant differences in all five features were found between normal and both precancerous and cancerous tissues. The only exception to this was a lack of statistically significant differences in the keratin fluorescence between normal tissues and papillary SCCs. Statistically significant differences were also observed in the epithelial thickness of dysplasia and carcinoma in situ, and in the keratin layer thickness of dysplasia and SCCs (both nonpapillary and papillary). This work clearly shows that three-dimensional images from MPLSM of endogenous tissue fluorescence can effectively distinguish between normal, precancerous, and cancerous epithelial tissues. This study provides the groundwork for further exploration into the application of multiphoton fluorescence endoscopy in a clinical setting.
We explored the role of Peyer's patch (PP) dendritic cell (DC) populations in the induction of immune responses to reovirus strain type 1 Lang (T1L). Immunofluorescence staining revealed the presence of T1L structural (sigma1) and nonstructural (sigmaNS) proteins in PPs of T1L-infected mice. Cells in the follicle-associated epithelium contained both sigma1 and sigmaNS, indicating productive viral replication. In contrast, sigma1, but not sigmaNS, was detected in the subepithelial dome (SED) in association with CD11c(+)/CD8alpha(-)/CD11b(lo) DCs, suggesting antigen uptake by these DCs in the absence of infection. Consistent with this possibility, PP DCs purified from infected mice contained sigma1, but not sigmaNS, and PP DCs from uninfected mice could not be productively infected in vitro. Furthermore, sigma1 protein in the SED was associated with fragmented DNA by terminal deoxy-UTP nick-end labeling staining, activated caspase-3, and the epithelial cell protein cytokeratin, suggesting that DCs capture T1L antigen from infected apoptotic epithelial cells. Finally, PP DCs from infected mice activated T1L-primed CD4(+) T cells in vitro. These studies show that CD8alpha(-)/CD11b(lo) DCs in the PP SED process T1L antigen from infected apoptotic epithelial cells for presentation to CD4(+) T cells, and therefore demonstrate the cross-presentation of virally infected cells by DCs in vivo during a natural viral infection.
Two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS) was used to investigate tumor-specific changes in the proteome of human colorectal cancers and adjacent normal mucosa. For each of six patients with different stages of colon cancer, Cy5-labeled proteins isolated from tumor tissue were combined with Cy3-labeled proteins isolated from neighboring normal mucosa and separated on the same 2-D gel along with a Cy2-labeled mixture of all 12 normal/tumor samples as an internal standard. Over 1500 protein spot-features were analyzed in each paired normal/tumor comparison, and using DIGE technology with the mixed-sample internal standard, statistically significant quantitative comparisons of each protein abundance change could be made across multiple samples simultaneously without interference due to gel-to-gel variation. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem (TOF/TOF) MS provided sensitive and accurate mass spectral data for database interrogation, resulting in the identification of 52 unique proteins (including redundancies due to proteolysis and post-translationally modified isoforms) that were changing in abundance across the cohort. Without the benefit of the Cy2-labeled 12 sample mixture internal standard, 42 of these proteins would have been overlooked due to the large degree of variation inherent between normal and tumor samples.
Intercellular signaling is essential for the development of teeth during embryogenesis and in maintenance of the continuously growing incisor teeth in postnatal rodents. WNT intercellular signaling molecules have been implicated in the regulation of tooth development, and the Wnt3 gene shows specific expression in the enamel knot at the cap stage. We demonstrate here that Wnt3 also is expressed in specific epithelial cell layers in postnatal incisor teeth. To begin to delineate the functions of Wnt3 in developing and postnatal teeth, we determined the effects of over- and ectopic expression of Wnt3 in the tooth epithelium of mice carrying a keratin 14-Wnt3 transgene. Expression of the transgene caused a progressive loss of ameloblasts from postnatal lower incisor teeth. Loss of ameloblasts may be due to defective proliferation or differentiation of ameloblast precursors, progressive apoptosis of ameloblasts, or loss of ameloblast stem cells.
BACKGROUND - It was recently shown that neuropilin-1 (NRP-1), which was described originally as a receptor for the semaphorins/collapsins (ligands involved in neuronal guidance), is a coreceptor for vascular endothelial growth factor (VEGF) and increases the affinity of specific isoforms of VEGF to its receptor, VEGF-R2.
METHODS - The authors investigated the expression and regulation of NRP-1 in human pancreatic adenocarcinoma specimens and cell lines.
RESULTS - Immunohistochemical analysis revealed that NRP-1 was expressed in 12 of 12 human pancreatic adenocarcinoma specimens but was absent in nonmalignant pancreatic tissue. Northern blot analysis revealed NRP-1 mRNA expression in 8 of 11 human pancreatic adenocarcinoma cell lines. NRP-1 mRNA expression was increased by epidermal growth factor (EGF) but not by tumor necrosis factor alpha in several of the human pancreatic adenocarcinoma cell lines studied. Treating human Panc-48 adenocarcinoma cells with EGF activated Akt and Erk but not P-38. Blockade of the phosphatidylinositol-3 kinase (PI-3K)/Akt, mitogen-activated protein kinase (MAPK)/Erk, or P-38 pathways abrogated EGF-induced NRP-1 expression. Finally, EGF receptor blockade in vivo led to a decrease in NRP-1 expression in an orthotopic model of human pancreatic carcinoma.
CONCLUSIONS - NRP-1 is expressed in most human pancreatic adenocarcinomas and cell lines but not in nonmalignant pancreatic tissue. EGF regulates NRP-1 expression through the PI-3K/Akt and MAPK/Erk signaling pathways, and blockade of the EGF receptor is associated with decreased expression of NRP-1 in vivo. NRP-1 may act as a coreceptor for VEGF in pancreatic carcinoma, as it does in other tumor systems, thereby enhancing angiogenesis and the effect of VEGF on the growth of pancreatic adenocarcinoma.
Copyright 2003 American Cancer Society.DOI 10.1002/cncr.11560
Mature alveolar type II cells that produce pulmonary surfactant are essential for adaptation to extrauterine life and prevention of infant respiratory distress syndrome. We have developed a new in vitro model to further investigate regulation of type II cell differentiation. Epithelial cells isolated from human fetal lung were cultured in serum-free medium on plastic. Cells treated with dexamethasone + cAMP analog and isobutylmethylxanthine for 4 days exhibited increased phosphatidylcholine synthesis and content of disaturated phosphatidylcholine species, manyfold increases in all surfactant proteins with processing to mature forms, and abundant lamellar bodies. DNA microarray analysis identified approximately 3,100 expressed genes, including subsets of genes induced 2- to >100-fold (approximately 2.5%) or repressed 2- to 18-fold (approximately 1.2%) by hormone treatment. Of the highly regulated genes, most were coregulated in an additive or synergistic manner by dexamethasone and cAMP agents. Approximately 90% of the regulated genes identified by this initial microarray analysis have not been previously recognized as hormone responsive. One newly identified hormone-induced gene is Nkx2.1 (thyroid transcription factor-1), which has a critical role in surfactant protein gene expression. Our findings indicate that glucocorticoid + cAMP is sufficient and necessary for precocious induction of functional type II cells in this in vitro system and that these hormones act primarily in combination to regulate expression of a subset of specific genes.
Lipoxygenase (LOX) metabolites from arachidonic acid and linoleic acid have been implicated in atherosclerosis, inflammation, keratinocyte differentiation and tumour progression. We previously showed that peroxisome proliferator-activated receptors (PPARs) play a role in keratinocyte differentiation and that the PPARalpha ligand 8S-hydroxyeicosatetraenoic acid is important in this process. We hypothesized that blocking LOX activity would block PPAR-mediated keratinocyte differentiation. Three LOX inhibitors, nordihydroguaiaretic acid, quercetin and morin, were studied for their effects on primary keratinocyte differentiation and PPAR activity. All three LOX inhibitors blocked calcium-induced expression of the differentiation marker keratin 1. In addition, activity of a PPAR-responsive element was inhibited in the presence of all three inhibitors, and this effect was mediated primarily through PPARalpha and PPARgamma. LOX inhibitors decreased the activity of a chimaeric PPAR-Gal4-ligand-binding domain reporter system and this effect was reversed by addition of PPAR ligands. Ligand-binding studies revealed that the LOX inhibitors bind directly to PPARs and demonstrate a novel mechanism for these inhibitors in altering PPAR-mediated gene expression.
Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.
Several members of the transforming growth factor beta (TGFbeta) superfamily are expressed in the developing murine epidermis. Among these are TGFbeta1, which is found in the basal layer, and bone morphogenetic protein (BMP)-6, located in the suprabasal layers. Although the role of TGFbeta in cell growth has been studied extensively, little is known about the effects of these factors on keratinocyte differentiation. This study demonstrates that BMP-6 acts to positively regulate the differentiation of primary skin keratinocytes grown in culture. In contrast, TGFbeta1 antagonizes keratinocyte differentiation blocking the upregulation of keratin markers by BMP-6. We show that the effects of BMP-6 on expression of keratin 1 (K1), a marker of differentiation, requires signaling through the Smad pathway. In addition, regulation of K1 levels by BMP-6 is modulated by the SEK signaling pathway. This suggests that regulation of keratinocyte differentiation by BMP-6 involves multiple signaling systems.
Copyright 2001 Academic Press.