MAB2058 Sigma-AldrichAnti-Integrin β4 Antibody, clone ASC-3

In adult tissues, multi-potent progenitor cells are some of the most primitive members of the developmental hierarchies that maintain homeostasis. That progenitors and their more mature progeny share identical genomes, suggests that fate decisions are directed by interactions with extrinsic soluble factors, ECM, and other cells, as well as physical properties of the ECM. To understand regulation of fate decisions, therefore, would require a means of understanding carefully choreographed combinatorial interactions. Here we used microenvironment protein microarrays to functionally identify combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells. Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell-cell versus cell-ECM interactions, as well as constellations of signaling molecules; and these were used in conjunction with physiologically relevant 3 dimensional human breast cultures. Both immortalized and primary human breast progenitors were analyzed. We report on the functional ability of those proteins of the mammary gland that maintain quiescence, maintain the progenitor state, and guide progenitor differentiation towards myoepithelial and luminal lineages.

MicroRNAs (miRs) are a novel class of RNAs with important roles in regulating gene expression. To identify miRs controlling prostate tumor progression, we utilized unique human prostate sublines derived from the parental P69 cell line, which differ in their tumorigenic properties in vivo. Grown embedded in laminin-rich extracellular matrix (lrECM) gels these genetically-related sublines displayed drastically different morphologies correlating with their behaviour in vivo. The non-tumorigenic P69 subline grew as multicellular acini with a defined lumen and basal/polar expression of relevant marker proteins. M12, a highly tumorigenic, metastatic derivative, grew as a disorganized mass of cells with no polarization, whereas the F6 subline, a weakly tumorigenic, non-metastatic M12 variant, reverted to acini formation akin to the P69 cell line. These sublines also differed in expression of vimentin, which was high in M12, but low in F6 and P69 sublines. Analysis of vimentin's conserved 3'-UTR suggested several miRs that could regulate vimentin expression. The lack of miR-17-3p expression correlated with an increase in vimentin synthesis and tumorigenicity. Stable expression of miR-17-3p in the M12 subline reduced vimentin levels 85% and reverted growth to organized, polarized acini in lrECM gels. In vitro motility and invasion assays suggested a decrease in tumorigenic behaviour, confirmed by reduced tumor growth in male athymic, nude mice dependent on miR-17-3p expression. Analysis of LCM-purified clinical human prostatectomy specimens confirmed that miR-17-3p levels were reduced in tumor cells. These results suggest that miR-17-3p functions as a tumor suppressor, representing a novel target to block prostate tumor progression.

Epithelial differentiation proceeds in at least two steps: Conversion of a nonepithelial cell into an epithelial sheet followed by terminal differentiation into the mature epithelial phenotype. It was recently discovered that the extracellular matrix (ECM) protein hensin is able to convert a renal intercalated cell line from a flat, squamous shape into a cuboidal or columnar epithelium. Global knockout of hensin in mice results in embryonic lethality at the time that the first columnar cells appear. Here, antibodies that either activate or block integrin beta1 were used to demonstrate that activation of integrin alpha v beta 1 causes deposition of hensin in the ECM. Once hensin polymerizes and deposits into the ECM, it binds to integrin alpha 6 and mediates the conversion of epithelial cells to a cuboidal phenotype capable of apical endocytosis; therefore, multiple integrins play a role in the terminal differentiation of the intercalated cell: alpha v beta 1 generates polymerized hensin, and another set of integrins (containing alpha 6) mediates signals between hensin and the interior of the cells.

We previously described the novel zinc finger protein ZKSCAN3 as a new "driver" of colon cancer progression. To investigate the underlying mechanism and because the predicted structural features (tandem zinc fingers) are often present in transcription factors, we hypothesized that ZKSCAN3 regulates the expression of a gene(s) favoring tumor progression. We employed unbiased screening to identify a DNA binding motif and candidate downstream genes. Cyclic amplification and selection of targets using a random oligonucleotide library and ZKSCAN3 protein identified KRDGGG as the DNA recognition motif. In expression profiling, 204 genes were induced 2-29-fold, and 76 genes reduced 2-5-fold by ZKSCAN3. To enrich for direct targets, we eliminated genes under-represented (less than 3) for the ZKSCAN3 binding motif (identified by CAST-ing) in 2 kilobases of regulatory sequence. Up-regulated putative downstream targets included genes contributing to growth (c-Met-related tyrosine kinase (MST1R), MEK2; the guanine nucleotide exchanger RasGRP2, insulin-like growth factor-2, integrin beta 4), cell migration (MST1R), angiogenesis (vascular endothelial growth factor), and proteolysis (MMP26; cathepsin D; PRSS3 (protease serine 3)). We pursued integrin beta 4 (induced up to 6-fold) as a candidate target because it promotes breast cancer tumorigenicity and stimulates phosphatidyl 3-kinase implicated in colorectal cancer progression. ZKSCAN3 overexpression/silencing modulated integrin beta 4 expression, confirming the array analysis. Moreover, ZKSCAN3 bound to the integrin beta 4 promoter in vitro and in vivo, and the integrin beta 4-derived ZKSCAN3 motif fused upstream of a tk-Luc reporter conferred ZKSCAN3 sensitivity. Integrin beta 4 knockdown by short hairpin RNA countered ZKSCAN3-augmented anchorage-independent colony formation. We also demonstrate vascular endothelial growth factor as a direct ZKSCAN3 target. Thus, ZKSCAN3 regulates the expression of several genes favoring tumor progression including integrin beta 4.

We have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates.

Human calcium-activated chloride channel 2 (hCLCA2) belongs to a family of multifunctional proteins and is localised mainly in basal cells of squamous epithelia. However, its function is still not fully understood. Relative amounts of hCLCA2 were analysed using real-time PCR in several human epithelial tissues and tissues expressing high amounts were identified. These tissues then underwent double immunolabelling with anti-hCLCA2 antibodies and antibodies against the adhesion molecules integrin beta4 and collagen VII and were visualised by fluorescence microscopy. Real-time PCR found hCLCA2 gene expression to be primarily associated with stratified squamous epithelia. Subsequent immunohistochemistry clearly demonstrated colocalisation between hCLCA2 and integrin beta4. This study reports on a possible underlying relationship between hCLCA2 and stratified epithelia and the close association of hCLCA2 with basal cell adhesion molecules in normal tissue, suggesting it may play an important role in basal cell attachment in stratified epithelia.

BACKGROUND: The generation of prostatic cell lines provides in vitro models for experimental studies of the pathogenesis of prostate carcinoma. Therefore, we established and characterized a new human prostate epithelial cell line, PEAZ-1 (prostate epithelial Arizona-1). METHODS: The PEAZ-1 cells were grown from a primary human prostate carcinoma specimen obtained from radical prostatectomy. The isolated cells were characterized by immunobiochemistry, immunohistochemistry, and tumorigenicity studies. RESULTS: PEAZ-1 cells are near diploid, tumorigenic, and androgen independent for cell growth. PEAZ-1 cells express N-cadherin, alpha- and beta-catenins, and p120 at cell-cell contacts, cytoplasmic laminin 5, vinculin, paxillin, and phosphotyrosine at focal adhesions, vimentin, and cytokeratins 8 and 18. They do not express plakoglobin, E-cadherin, and PSA, and do not form desmosomes and hemidesomomes. PEAZ-1 respond to ocadaic acid, a pro-apoptotic agent, by expression of p53. CONCLUSIONS: PEAZ-1 cells is a human prostate cancer cell line that has a number of mesenchymal characteristics.