Correlation between E-cadherin interactions, survivin expression, and apoptosis in MDCK and ts-Src MDCK cell culture models.

Survivin, a member of inhibitor of apoptosis (IAP) protein family, is a multifunctional protein expressed in most cancers. In addition to inhibition of apoptosis, it regulates proliferation and promotes migration. Its presence and function in cells is strongly regulated via transcription factors, intracellular localization, and degradation. We analyzed the presence of survivin at protein level in various culture environments and under activation of Src tyrosine kinase in epithelial canine kidney MDCK cells in order to elucidate factors controlling survivin 'lifespan'. We used untransformed and temperature sensitive ts-Src MDCK cells as a model and forced them to grow in suspension (1D), in 2D on hard and soft surfaces and in soft 3D Matrigel environment with or without EGTA. In addition, we tested the effect of stressful conditions by cultivating the cells in the presence of an anti-cancer drug and a generator of reactive oxygen species (ROS), piperlongumine (PL) with or without an antioxidant, N-acetylcysteine (NAC). We could confirm that inhibition of apoptosis and simultaneous downregulation of survivin in MDCK cells required both intact cell-cell junctions, trans-interactions of E-cadherin and soft 3D matrix environment. In ts-Src-transformed MDCK cells, survivin was upregulated as soon as the cell-cell junctions were disintegrated. ROS generation with PL-induced cell death of ts-Src MDCK cells concomitantly with survivin downregulation. NAC rescued the ts-Src MDCK cells from ROS-induced apoptosis without upregulation of survivin resulting in a situation resembling untransformed MDCK cells in 3D environment and E-cadherin delineating the lateral cell walls.

MDCK cells are capable of water secretion and reabsorption in response to changes in the ionic environment.

A prerequisite for tissue electrolyte homeostasis is highly regulated ion and water transport through kidney or intestinal epithelia. In the present work, we monitored changes in the cell and luminal volumes of type II Madin-Darby canine kidney (MDCK) cells grown in a 3D environment in response to drugs, or to changes in the composition of the basal extracellular fluid. Using fluorescent markers and high-resolution spinning disc confocal microscopy, we could show that lack of sodium and potassium ions in the basal fluid (tetramethylammonium chloride (TMACl) buffer) induces a rapid increase in the cell and luminal volumes. This transepithelial water flow could be regulated by inhibitors and agonists of chloride channels. Hence, the driving force for the transepithelial water flow is chloride secretion, stimulated by hyperpolarization. Chloride ion depletion of the basal fluid (using sodium gluconate buffer) induces a strong reduction in the lumen size, indicating reabsorption of water from the lumen to the basal side. Lumen size also decreased following depolarization of the cell interior by rendering the membrane permeable to potassium. Hence, MDCK cells are capable of both absorption and secretion of chloride ions and water; negative potential within the lumen supports secretion, while depolarizing conditions promote reabsorption.

Lysyl hydroxylase 3 is secreted from cells by two pathways.

Lysyl hydroxylase 3 (LH3) is a post-translational modification enzyme with lysyl hydroxylase (LH), collagen galactosyltransferase (GT), and glucosyltransferase (GGT) activities. The active sites responsible for LH and GT/GGT activities of LH3 are localized separately in the carboxy- and the amino-terminal parts of the molecule, respectively. LH3 is found both intracellularly in the ER, as well as extracellularly in serum, the extracellular space and on cell surfaces, and is the only secreted LH isoform. In order to determine whether the activities of LH3 play a role in the secretion, we created various LH3 and mutant expression constructs and over-expressed the proteins in COS-7 and HT-1080 cells. Our data indicate that while the LH active site mediates retention of LH3 in the ER, the GGT active site is required for the secretion of LH3 into the extracellular space. Moreover, Brefeldin A treatment and cholesterol depletion of the cells revealed that the secretion of LH3 from the ER to the extracellular space occurs via two secretory pathways, which generate two glycoforms. LH3 molecules found in the cell medium are secreted through the Golgi complex, and the secretion is dependent on LH3 glycosyltransferase activity. LH3 found on the cell surface bypasses the Golgi complex.

Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays.

Differentiation and transformation of untransformed and ts-Src-transformed canine kidney MDCK cells in 2D and 3D environment were investigated using microarray technique, RT-PCR, confocal microscopy and functional assays. Activated Src induced epithelial-mesenchymal transition (EMT) in 2D environment followed by translocation of junctional proteins to the cytoplasm, without significant changes in protein expression. In 3D environment untransformed MDCK cells formed cell cysts with apical domain facing a lumen, E-cadherin delineating the lateral membranes, ZO-1 at tight junctions and caspase-3 in apoptotic cells captured within the lumen. This was accompanied by reduced expression of an apoptosis inhibitor, survivin and vesicle transport effectors, rab 7 and 8, whereas rab 5 expression increased. In 3D environment activated Src induced changes in expression of over 100 genes as revealed by microarray analysis, mostly involved in cell signaling, division and energy metabolism. Only response in cytoskeletal components was decreased expression of actin and Arp2/3 by v-Src, whereas two p120catenin binding proteins Kaiso and Nanos increased their expression. Concomitantly, apoptosis was inhibited by v-Src resulting in formation of a sphere with epitheloid cells facing extracellular matrix and undifferentiated cells captured within the cluster. This was accompanied by increased expression of apoptosis inhibitor survivin, as revealed by western blotting. Mitochondrial membrane potential in untransformed MDCK cells was lower than in ts-Src-MDCK cells in early days of cluster formation correlating with the induction of apoptosis. Hence, v-Src activation in 3D environment did not induce EMT, but brought about inhibition of apoptosis and increased proliferation where increased expression of survivin and inhibition of the mitochondrial permeability have a role.

The glycosyltransferase activities of lysyl hydroxylase 3 (LH3) in the extracellular space are important for cell growth and viability.

Lysyl hydroxylase (LH) isoform 3 is a post-translational enzyme possessing LH, collagen galactosyltransferase (GT) and glucosyltransferase (GGT) activities. We have demonstrated that LH3 is found not only intracellularly, but also on the cell surface and in the extracellular space, suggesting additional functions for LH3. Here we show that the targeted disruption of LH3 by siRNA causes a marked reduction of both glycosyltransferase activities, and the overexpression of LH3 in HT-1080 cells increases hydroxylation of lysyl residues and the subsequent galactosylation and glucosylation of hydroxylysyl residues. These data confirm the multi-functionality of LH3 in cells. Furthermore, treatment of cells in culture medium with a LH3 N-terminal fragment affects the cell behaviour, rapidly leading to arrest of growth and further to lethality if the fragment is glycosyltransferase-deficient, and leading to stimulation of proliferation if the fragment contains LH3 glycosyltransferase activities. The effect is reversible, the cells recovering after removal of the glycosyltransferase-deficient fragment. The findings were confirmed by overexpressing the full-length LH3 in native or mutated forms in the cells. The data indicate that the increase in proliferation depends on the glycosyltransferase activity of LH3. The overexpression of a glycosyltransferase-deficient mutant or targeted disruption of LH3 by siRNA in cells results in abnormal cell morphology followed by cell death. Our data clearly indicate that the deficiency of LH3 glycosyltransferase activities, especially in the extracellular space, causes growth arrest revealing the importance of the glycosyltransferase activities of LH3 for cell growth and viability, and identifying LH3 as a potential target for medical applications, such as cancer therapy.

KRAS and BRAF mutations induce anoikis resistance and characteristic 3D phenotypes in Caco­2 cells.

In a number of types of cancer, anoikis, a form of apoptosis induced by loss of extracellular matrix (ECM) attachment, is disturbed. Anoikis resistance is essential in the formation of metastases. A recent study identified carcinoma cell subpopulations surviving without ECM contact in pathological specimens of colorectal cancer. The occurrence of these subpopulations indicated anoikis resistance. In the present study, it is demonstrated that KRAS and BRAF mutations induce anoikis resistance in colon cancer (Caco­2) cells. In 3D cultures, Caco­2 cells transfected with mutated KRAS or BRAF formed multicellular structures analogous to anoikis­resistant subpopulations in actual carcinomas, and serve as an in vitro model for anoikis resistance. Caco­2 cell lines were constructed, with KRAS or BRAF mutations, using retroviral delivery. The current study investigated anoikis resistance using an Annexin V apoptosis test from suspension cultures. 3D in vitro cultures, which were generated in collagen­matrigel mixtures, were assessed using confocal microscopy. 3D cultures embedded in paraffin were analyzed using conventional histopathology. In suspension cultures, Caco­2 cells with KRAS or BRAF mutations indicated a significantly lower proportion of Annexin positivity than the native Caco­2 cells, indicating that these mutations induce anoikis resistance in Caco­2 cells. 3D cultures displayed native Caco­2 cells forming polarized cysts with a single layer thick epithelium, whereas Caco­2 cells with KRAS or BRAF mutations formed partially filled cystic structures or solid round structures where only the outermost layer was in contact with the ECM. Additionally, KRAS mutations induced reversed polarity to Caco­2 cells along with the emergence of solid growth. The present study demonstrated that KRAS and BRAF mutations induce anoikis resistance in Caco­2 colorectal cancer cells. The growth patterns generated from the KRAS and BRAF mutated cells in 3D cultures revealed a resemblance to the putative anoikis­resistant subpopulations in actual carcinomas, including micropapillary structures and solid tumor cell islands. Additionally, KRAS mutation induced the emergence of inverted polarity. In conclusion, 3D cultures with modified Caco­2 cells serve as a valid in vitro model for anoikis resistance and inverted polarity.

Micropapillary Structures in Colorectal Cancer: An Anoikis-resistant Subpopulation.

BACKGROUND/AIM: Micropapillary structures (MIPs) are focal piles of columnar cells without extracellular matrix contact, and common in serrated colorectal carcinoma (CRC). In order to characterize biology of MIPs in colorectal cancer (CRC), the proliferation and apoptosis rates, and survivin expression were compared between MIPs and other cancer epithelial cells of CRC (non-MIPs). MATERIALS AND METHODS: We assessed 46 samples of normal colorectal mucosa, 62 carcinomas and 54 polyps for proliferation (Ki67), apoptosis (M30), and survivin expression by immunohistochemistry. RESULTS: MIPs in carcinoma showed lower rates of proliferation and apoptosis than non-MIPs. A low rate of apotosis in MIPs was associated with poor prognosis in local carcinoma. In normal crypts, nuclear-to-cytoplasmic transition of survivin indicated epithelial cell maturation. Cancer cases showed increased cytoplasmic expression of survivin than normal mucosa and polyps. However, MIPs showed lower nuclear and cytoplasmic survivin expression than non-MIPs. Our findings suggest that MIPs represent a biologically distinct subpopulation of carcinoma cells with features of anoikis resistance and possibly quiescence.

Decoy oligonucleotide characterization of GATA-4 transcription factor in hypertrophic agonist induced responses of cardiac myocytes.

GATA-4 transcription factor is required for normal cardiac development. However, it is unknown whether GATA-4 is an essential mediator of hypertrophic responses in the heart. Rat B-type natriuretic peptide (BNP) gene promoter contains a region of two adjacent GATA binding sites (between -68 and -97) with high affinity for GATA-4. In order to block GATA-4 dependent signaling in cultured neonatal rat ventricular myocytes we administered a synthetic 30-bp phosphorothioated double-stranded DNA complementary to the rat BNP promoter region (between -68 and -97) as a "decoy" cis-element to bind GATA-4. GATA decoy oligodeoxynucleotide treatment of cardiomyocytes blocked GATA-4 DNA binding activity in electrophoretic mobility shift analysis and decreased baseline expression of cardiac natriuretic peptides and GATA-dependent promoter activity. In contrast, blocked GATA-4 DNA binding did not prevent endothelin-1 or phenylephrine induced expression of cardiac natriuretic peptides. Mutation of GATA binding sites at -80 and -91 rat BNP promoter downregulated baseline but did not affect endothelin-1 or angiotensin II induced promoter activity. Additively, GATA decoy oligodeoxynucleotide treatment was insufficient to block endothelin-1 induced activation of protein synthesis or sarcomeric protein assembly. In conclusion, a targeted disruption of GATA-4 DNA binding activity is insufficient to prevent hypertrophic agonist induced responses of ventricular myocytes.

Cofilin regulator 14-3-3zeta is an evolutionarily conserved protein required for phagocytosis and microbial resistance.

Phagocytosis is an ancient cellular process that plays an important role in host defense. In Drosophila melanogaster phagocytic, macrophage-like hemocytes recognize and ingest microbes. We performed an RNAi-based in vitro screen in the Drosophila hemocyte cell line S2 and identified Abi, cpa, cofilin regulator 14-3-3ζ, tlk, CG2765, and CG15609 as mediators of bacterial phagocytosis. Of these identified genes, 14-3-3ζ had an evolutionarily conserved role in phagocytosis: bacterial phagocytosis was compromised when 14-3-3ζ was targeted with RNAi in primary Drosophila hemocytes and when the orthologous genes Ywhab and Ywhaz were silenced in zebrafish and mouse RAW 264.7 cells, respectively. In Drosophila and zebrafish infection models, 14-3-3ζ was required for resistance against Staphylococcus aureus. We conclude that 14-3-3ζ is essential for phagocytosis and microbial resistance in insects and vertebrates.

SRC-induced disintegration of adherens junctions of madin-darby canine kidney cells is dependent on endocytosis of cadherin and antagonized by Tiam-1.

The effects of Src tyrosine kinase activation in subconfluent temperature sensitive (ts)-Src-transformed Madin-Darby canine kidney (MDCK) cells were analyzed by shifting them from nonpermissive (40.5 degrees C) to permissive (35 degrees C) temperature. Already, in 15 minutes, adherens junction components were released from the lateral walls and accumulated to basal surfaces. Simultaneously, membranous actin staining vanished, actin bundles appeared at the basal surface, and the cells flattened. The only component phosphorylated and translocated after the shift to 35 degrees C was p120ctn. The epithelial-mesenchymal transition could be inhibited by a specific inhibitor of Src kinase, PP2, or by inhibiting endocytosis. Therefore, Src activation was responsible for the transition, but not because of phosphorylation of adherens junction components but by way of activation of endocytic machinery and RhoGTPase. Expression of an RacGEF, Tiam-1 (T-lymphoma invasion and metastasis gene 1), prevented flattening of Src-transformed MDCK cells at 35 degrees C and resulted in accumulation of cadherin to lateral membranes. In the case where the Src-MDCK cells were cultivated at 35 degrees C and shifted for short time periods to 40.5 degrees C, cadherin rapidly returned to lateral membranes, whereas actin and p120ctn followed hours afterward. This further supports the view that cadherin internalization is the primary target of Src kinase. We also looked at the cell morphology and distribution of cadherin and Tiam-1 in cells grown in three-dimensional gels composed of collagen and laminin or in Matrigel. At nonpermissive temperature, both Src-MDCK and Tiam-1-transfected Src-MDCK cells exhibited nonpolarized morphology in collagen I, a loose cluster in the mixture of collagen I and laminin, and a differentiated cyst in Matrigel. In growth factor-depleted Matrigel, the Src-MDCK cells grew in nondifferentiated clusters, whereas Tiam-1-transfected cells went to apoptosis. The differentiated phenotype of both cell lines could be rescued by Matrigel-conditioned medium, platelet-derived growth factor, or cholera toxin. Concomitantly, both cadherin and Tiam-1 were recruited to lateral membranes. Therefore, cadherin and Tiam-1 seem to be the key players in the differentiation process of MDCK cells.

Membrane potential and endocytic activity control disintegration of cell-cell adhesion and cell fusion in vinculin-injected MDBK cells.

Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961-974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell-cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin-membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin-membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local and transient pH gradients play a role.

Apolipoprotein E and colon cancer. Expression in normal and malignant human intestine and effect on cultured human colonic adenocarcinoma cells.

Background: Apolipoprotein E (apo E) is a key regulatory protein in lipoprotein metabolism and it is also a potent inhibitor of cell proliferation. Although genetic alterations of apo E affect enterohepatic cholesterol transport and, presumably, the risk of colon carcinoma, the expression and potential functions of apo E in the human intestine are poorly known. Methods: The localization of apo E in normal and malignant gastrointestinal tract was studied using immunohistochemistry and in situ hybridization. The effect of apo E3 on cell polarity and the distribution of beta-catenin was examined in HT29 human colon adenocarcinoma cell lines. Results: Both apo E protein and mRNA were present throughout human intestine. The macrophages in the superficial lamina propria of normal colon were more strongly positive for apo E than those in the small intestine, where the most positively stained cells were dendritic cells and macrophages in the germinal centers of lymphoid follicles. In carcinomas, intensely positive macrophages surrounded the tumor area. In cultured undifferentiated HT29 cells, treatment with apo E improved cell polarity and translocated beta-catenin from the cytoplasm to cell--cell adhesion sites. Conclusions: Mononuclear phagocytes and endocrine cells are the main source of apo E in the gastrointestinal tract. We hypothesize that macrophage-derived apo E may modulate epithelial integrity and thus contribute to cell growth.