calcein-am and Adenocarcinoma

Multidrug resistance-associated protein 2 (MRP2, ABCC2) is the second member of the MRP transporter family and functions physiologically as an organic anion transporter. Earlier studies have confirmed that radixin, which is a member of the ERM (ezrin/radixin/moesin) family, modulates MRP2 localization at the canalicular membrane in hepatocytes. The relationship between radixin and MRP2 - particularly, the effect of radixin on the expression and function of MRP2 in cells or tissues that co-express all three ERM proteins - has not been well studied. To examine the role of radixin in the expression and function of MRP2 and other MRPs, we chose human gastric carcinoma SGC-7901 cells that express all three ERM proteins rather than hepatocytes, which predominantly express radixin. Radixin stable knockdown SGC-7901 cells, which were constructed by RNAi, exhibited no compensatory up-regulation of ezrin or moesin. The mRNA expression profiles of MRPs in the radixin knockdown cells were primarily evaluated by RT-PCR. Real time quantitative RT-PCR and western blot analysis revealed that the radixin deficiency caused the mRNA and protein expression levels of MRP2 to be reduced by about 50%, respectively. Accordingly, efflux and MTT assays showed that the radixin knockdown cells exhibited lower efflux ability with respect to calcein but no significant change in cell viability. In conclusion, among the MRP1-6 family members, radixin selectively modulates the expression and function of MRP2 in a system co-expressing all three ERM proteins.

Topics: Adenocarcinoma; Biological Transport; Blotting, Western; Cell Line, Tumor; Cell Survival; Cytoskeletal Proteins; Fluoresceins; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Membrane Proteins; Microfilament Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Stomach Neoplasms

Putative cancer stem cell (CSC) populations efflux dyes such as Hoechst 33342 giving rise to side populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoechst 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non-toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (C(lo)P). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress and proliferation in several cancer cell lines when stained whereas Hoechst 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53(-/-) cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.

Topics: Adenocarcinoma; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Apoptosis; ATP-Binding Cassette Transporters; bcl-2-Associated X Protein; Benzimidazoles; Benzothiazoles; Biological Transport, Active; Calcium Channel Blockers; Cell Line, Tumor; Colonic Neoplasms; DNA Damage; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Genes, p53; Humans; Isoenzymes; Microscopy, Fluorescence; Neoplasm Proteins; Neoplastic Stem Cells; Prodrugs; Retinal Dehydrogenase; Staining and Labeling; Tumor Suppressor Protein p53

Topics: Adenocarcinoma; ATP-Binding Cassette Transporters; Benzimidazoles; Biological Transport, Active; Cell Line, Tumor; Cell Separation; Colonic Neoplasms; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Neoplasm Proteins; Neoplastic Stem Cells; Phenotype; Prodrugs; Staining and Labeling

Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however, these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12h. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch.

Topics: Adenocarcinoma; Animals; Biomechanical Phenomena; Cell Culture Techniques; Cell Line; Cell Line, Tumor; Computer Simulation; Endothelial Cells; Endothelium, Vascular; Epithelial Cells; Finite Element Analysis; Fluoresceins; Fluorescent Dyes; Humans; Lung Neoplasms; Mechanotransduction, Cellular; Mice; Myoblasts; Skin; Substrate Specificity

Activated macrophages (M phi) found in the intestinal lesions of patients with inflammatory bowel disease (IBD) secrete many inflammatory mediators which can regulate intestinal epithelial cell (IEC) function. However, little is known about direct M phi-IEC interactions. Two potential mechanisms by which cells may interact are through specific receptor-ligand binding of adhesion molecules, such as integrins or cadherins, and by exchange of cytoplasmic substances through transmembraneous channels called gap junctions. We investigated whether M phi could adhere to epithelial cells in culture and form transmembrane communication channels as defined by dye transfer. Primary cultures of murine M phi and a M phi cell line, P388D1, adhered to Mode-K and IEC6, but not CMT-93 IEC. Antibody blocking studies determined that P388D1-Mode-K binding was partially dependent on beta 2 integrin (CD18) function, Mode-K constitutively expressed CD106 (VCAM-1) and cell associated fibronectin, while P388D1 expressed low levels of CD49d/CD29 (VLA4) but blocking antibodies to these surface molecules did not inhibit P388D1-Mode-K adherence. Transfer of calcein dye from M phi to IEC was quantitated by flow cytometry and was dependent on M phi-IEC adhesion. Dye transfer was concentration dependent in that the fluorescence intensity of Mode-K was proportional to the number of adherent P388D1 cells as well as the dye load of the M phi. These results indicate that M phi interact with IEC by adhesion and possibly through gap junctions and may thus regulate IEC function by direct cell-cell communication.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Biological Transport; Bone Marrow Cells; CD18 Antigens; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cells, Cultured; Connexin 43; Epithelial Cells; Female; Fluoresceins; Fluorescent Dyes; Intestinal Mucosa; L Cells; Macrophages; Macrophages, Peritoneal; Mice; Mice, Inbred C3H; Rats; Rectal Neoplasms; Tumor Cells, Cultured