Bernard W Futscher

The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood.

PMID: 14670180;PMCID: PMC1502613;Abstract:

The maspin gene is not expressed in normal human pancreas, but its expression is acquired during human pancreatic carcinogenesis. In other normal human cells and their malignant counterparts, maspin expression is controlled through the epigenetic state of its promoter. In studies presented herein, we used bisulfite genomic sequencing and chromatin immunoprecipitation studies to show that maspin-negative pancreas cells have a methylated maspin promoter, and that the associated H3 and H4 histones are hypoacetylated. In contrast to normal pancreas, four of six human pancreatic carcinoma cell lines investigated displayed activation of maspin expression. This activation of maspin expression in pancreatic carcinoma cells was linked to demethylated promoters and hyperacetylation of the associated H3 and H4 histones. In addition, 5-aza-2′-deoxycytidine treatments activated maspin expression in the two maspin-negative pancreatic carcinoma cell lines, suggesting a causal role for cytosine methylation in the maintenance of a transcriptionally silent maspin gene. Thus, human pancreatic carcinoma cells acquire maspin expression through epigenetic derepression of the maspin locus, and in so doing appear to co-opt a normal cellular mechanism for the regulation of this gene.

PMID: 7513198;Abstract:

In contrast to its clearly defined role as a multidrug efflux pump in neoplastic cells, the physiologic function of P-glycoprotein (P-gly) in normal cells is unclear. Recent reports identifying P-gly in normal blood and bone marrow suggest that hematopoietic development or function may be dependent on P-gly. To understand the normal function of P-gly in the blood, its level of expression and function must first be quantitated relative to a known standard. In this study, P-gly, MDR1 gene expression, and P-gly function were quantitated in normal leukocytes. P-gly and MDR1 expression were analyzed in individual leukocyte lineages (T-helper, T-suppressor, monocyte, granulocyte, B-lymphocyte, NK cell) from normal volunteers. P-gly on the cell surface was detected by fluorescent double-labeling for lineage (CD4, CD8, CD14, CD15, CD19, CD56, respectively) and P-gly (MRK16) with analysis by flow cytometry and in some cases immunoblot analysis. MDR1 mRNA analysis on purified lineages was performed using quantitative reverse transcription-polymerase chain reaction. P-gly function was determined for each lineage using dual-labeling for lineage and P-gly substrate (rhodamine 123). The P-gly expressing human myeloma cell line, 8226/Dox6, was used as a reference of comparison for levels of P-gly, MDR1 mRNA, and function. CD56⁺ cells expressed the highest levels of MDR1 mRNA followed by CD8⁺ > CD4⁺ ≃ CD15⁺ > CD19⁺ > CD14⁺, with percentage values relative to Dox6 of 49%, 17%, 8%, 8%, 4%, and 2%, respectively. The assays for P-gly immunofluorescence and function correlated well with mRNA analysis except for CD15⁺ cells (granulocytes), which showed a moderate MDR1 mRNA level with a lack of both function and surface P-gly staining. Granulocyte membranes did show P-gly on immunoblot analysis when probed with either C219 or JSB1. We conclude that (1) P-gly and the MDR1 mRNA are expressed in normal leukocytes, (2) this P-gly expression is lineage specific with relatively high levels among CD56⁺ cells, and (3) the expression of P-gly in granulocytes is not associated with transport of the P-gly substrate, rhodamine 123, out of the cell.