Chromatin structure of the regulatory regions of pS2 and cathepsin D genes in hormone-dependent and -independent breast cancer cell lines

We have compared the DNase I hypersensitivity of the regulatory region of two estrogen-regulated genes, pS2 and cathepsin D in hormone-dependent and -independent breast carcinoma cell lines. This strategy allowed the identification of two important control regions, one in pS2 and the other in cathepsin D genes. In the hormone-dependent MCF7 cell line, within the pS2 gene 5'-flanking region, we detected two major DNase I hypersensitive sites, induced by estrogens and/or IGFI: pS2-HS1, located in the proximal promoter and pS2-HS4, located -10.5 Kb from the CAP site, within a region that has not been cloned. The presence of these two DNase I hypersensitive sites correlates with pS2 expression. Interestingly in MCF7 cells, estrogens and IGFI induced indistinguishable chromatin structural changes over the pS2 regulatory region, suggesting that the two transduction-pathways converge to a unique chromatin target. In two cell lines that do not express pS2, MDA MB 231, a hormone-independent cell line that lacks the estrogen receptor alpha, and HE5, a cell line derived from MDA MB 231 by transfection that expresses estrogen receptor alpha, there was only one hormone-independent DNase I hypersensitive site. This site, pS2-HS2, was located immediately upstream of pS2-HS1. In MCF7 cells, two major DNase I hypersensitive sites were present in the 5'-flanking sequences of the cathepsin D gene, which is regulated by estrogens in these cells. These sites, catD-HS2 and catD-HS3, located at positions -2.3 Kb and -3.45 Kb, respectively, were both hormone-independent. A much weaker site, catD-HS1, covered the proximal promoter. In MDA MB 231 cells, that express cathepsin D constitutively, we detected an additional strong hormone-independent DNase I hypersensitive site, catD-HS4, located at position -4.3 Kb. This region might control the constitutive over-expression of cathepsin D in hormone-independent breast cancer cells. All together, these data demonstrate that a local reorganization of the chromatin structure over pS2 and cathepsin D promoters accompanies the establishment of the hormone-independent phenotype of the cells.     

Secretion of plasminogen activator inhibitor 2 by human peripheral blood monocytes occurs via an endoplasmic reticulum-golgi-independent pathway

Plasminogen activator inhibitor 2 (PAI-2) is a serine protease inhibitor (serpin) that is secreted and accumulated intracellularly by monocytes. We investigated PAI-2 synthesis by isolated human peripheral blood monocytes and found that a 47-kDa nonglycosylated form of PAI-2 was abundant in conditioned medium from monocytes. Secretion of PAI-2 by monocytes was not inhibited by agents that inhibit either ER-Golgi pathway-dependent secretion, brefeldin A, or N-linked glycosylation, tunicamycin. IL-1beta served as a control for a protein that is secreted by an ER-Golgi-independent pathway, and secretion of IL-1beta was not inhibited by brefeldin A. This was in contrast to secretion of TNFalpha, which was dependent on the ER-Golgi pathway. None of the treatments was cytotoxic toward monocytes, as measured by release of the intracellular enzyme lactate dehydrogenase (LDH) into the conditioned medium. Subcellular fractionation revealed that PAI-2 and IL-1beta were colocalized. The mechanism for secretion of PAI-2 was not dependent on calcium or intracellular trafficking via the classical vesicular mechanism(s), distinguishing it from IL-1beta secretion. These studies show that PAI-2 is secreted by primary human monocytes via an ER-Golgi-independent pathway.