Novel senescence associated gene, YPEL3, is repressed by estrogen in ER+ mammary tumor cells and required for tamoxifen-induced cellular senescence.

Estrogen signaling plays an important role in breast carcinogenesis. An increased understanding of estrogen gene targets and their effects will allow for more directed and effective therapies for individuals with breast cancer, particularly those with estrogen receptor positive tumors resistant to tamoxifen therapy. Here, we identify YPEL3 as a growth suppressive protein downregulated by estrogen in estrogen receptor positive breast cancer cell lines. Estrogen repression of YPEL3 expression was found to be independent of p53 but dependent on estrogen receptor alpha expression. Importantly, YPEL3 expression, which is induced by the removal of estrogen or treatment with tamoxifen triggers cellular senescence in MCF-7 cells while loss of YPEL3 increases the growth rate of MCF-7 cells. Taken together these findings suggest that YPEL3 may represent a potential target for directed hormonal therapy for estrogen receptor positive breast cancer patients.

Senescence-associated gene YPEL3 is downregulated in human colon tumors.

BACKGROUND: Previous work has demonstrated YPEL3 to be a growth-suppressive protein that acts through a pathway of cellular senescence. We set out to determine whether human colon tumors demonstrated downregulation of YPEL3. METHODS: We collected colon tumor samples with matched normal control samples and analyzed them for YPEL3 gene expression by reverse transcriptase-polymerase chain reaction and CpG hypermethylation of the YPEL3 promoter by base-specific polymerase chain reaction analysis. Colon cancer cell lines (Caco-2 and HCT116(-/-) p53) were used to assess YPEL3 gene expression after treatment with 5-azadeoxycytidine or trichostatin A. RESULTS: Reverse transcriptase-polymerase chain reaction analysis demonstrated a decrease in YPEL3 expression in tumor samples when compared to their patient-matched normal tissue. We determined that DNA methylation of the YPEL3 promoter CpG island does not play a role in YPEL3 regulation in human colon tumors or colon cancer cells lines, consistent with the inability of 5-azadeoxycytidine treatment to induce YPEL3 expression in colon cancer cell lines. In contrast, colon cell line results suggest that histone acetylation may play a role in YPEL3 regulation in colon cancer. CONCLUSIONS: YPEL3 is preferentially downregulated in human colon adenocarcinomas. DNA hypermethylation does not appear to be the mechanism of YPEL3 downregulation in this subset of collected patient samples or in colon cell lines. Histone acetylation may be a relevant epigenetic modulator of YPEL3 in colon adenocarcinomas. Future investigation of YPEL3 and its role in colon cancer signaling and development may lead to increased understanding and alternative treatment options for this disease.

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages.

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.

Identification and structural ramifications of a hinge domain in apolipoprotein A-I discoidal high-density lipoproteins of different size.

Apolipoprotein (apo) A-I is the major protein constituent of human high-density lipoprotein (HDL) and is likely responsible for many of its anti-atherogenic properties. Since distinct HDL size subspecies may play different roles in interactions critical for these properties, a key question concerns how apoA-I can adjust its conformation in response to changes in HDL particle size. A prominent hypothesis states that apoA-I contains a flexible "hinge domain" that can associate/dissociate from the lipoprotein as its diameter fluctuates. Although flexible domains clearly exist within HDL-bound apoA-I, this hypothesis has not been directly tested by assessing the ability of such domains to modulate their contacts with the lipid surface. In this work, discoidal HDL particles of different size were reconstituted with a series of human apoA-I mutants containing a single reporter tryptophan residue within each of its 22 amino acid amphipathic helical repeats. The particles also contained nitroxide spin labels, potent quenchers of tryptophan fluorescence, attached to the phospholipid acyl chains. We then measured the relative exposure of each tryptophan probe with increasing quencher concentrations. We found that, although there were modest structural changes across much of apoA-I, only helices 5, 6, and 7 exhibited significant differences in terms of exposure to lipid between large (96 A) and small (78 A) HDL particles. From these results, we present a model for a putative hinge domain in the context of recent "belt" and "hairpin" models of apoA-I structure in discoidal HDL particles.

Helix orientation of the functional domains in apolipoprotein e in discoidal high density lipoprotein particles.

Human apolipoprotein E (apoE) mediates high affinity binding to the low density lipoprotein receptor when present on a lipidated complex. In the absence of lipid, however, apoE does not bind the receptor. Whereas the x-ray structure of lipid-free apoE3 N-terminal (NT) domain is known, the structural organization of its lipid-associated, receptor-active conformation is poorly understood. To study the organization of apoE amphipathic alpha-helices in a lipid-associated state, single tryptophan-containing apoE3 variants were employed in fluorescence quenching studies. The relative positions of the Trp residues with respect to the phospholipid component of apoE/lipid particles were established from the degree of quenching by phospholipids bearing nitroxide groups at various positions along their fatty acyl chains. Four apoE3-NT variants bearing Trp reporter groups at positions 141, 148, 155, or 162 within helix 4 and two apoE3 variants containing single Trp at positions 257 or 264 in the C-terminal (CT) domain, were reconstituted into phospholipid-containing discoidal complexes. Parallax analysis revealed that each engineered Trp residue in helix 4 of apoE3-NT, as well as those in the CT domain of apoE, localized approximately 5 A from the center of the bilayer. Circular dichroism studies revealed that lipid association induces additional helix formation in apoE. Protease protection assays suggest the flexible loop segment between the NT and CT domains may transition from unstructured to helix upon lipid association. Taken together, these data support a model wherein the alpha-helices in the receptor-binding region and the CT domain of apoE align perpendicular to the fatty acyl chains of the phospholipid bilayer. In this alignment, the residues of helix 4 are arrayed in a positively charged, curved helical segment for optimal receptor interaction.

Ceramide enhances cholesterol efflux to apolipoprotein A-I by increasing the cell surface presence of ATP-binding cassette transporter A1.

It is widely accepted that functional ATP-binding cassette transporter A1 (ABCA1) is critical for the formation of nascent high density lipoprotein particles. However, the cholesterol pool(s) and the cellular signaling processes utilized by the ABCA1-mediated pathway remain unclear. Sphingomyelin maintains a preferential interaction with cholesterol in membranes, and its catabolites, especially ceramide, are potent signaling molecules that could play a role in ABCA1 regulation or function. To study the potential role of ceramide in this process, we treated a variety of cell lines with 20 microM C2-ceramide and examined apolipoprotein-mediated cholesterol efflux to lipid-free apoA-I. We found that cell lines expressing ABCA1 displayed 2-3-fold increases in cholesterol efflux to apoA-I. Cell lines not expressing ABCA1 were unaffected by ceramide. We further characterized the cholesterol efflux effect in Chinese hamster ovary cells. Ceramide treatment did not cause significant cytotoxicity or apoptosis and did not affect cholesterol efflux to non-apolipoprotein acceptors. Raising endogenous ceramide levels increased cholesterol efflux to apoA-I. Using a cell surface biotinylation method, we found that the total cellular ABCA1 and that at the plasma membrane were increased with ceramide treatment. Also ceramide enhanced the binding of fluorescently labeled apoA-I to Chinese hamster ovary cells. These data suggest that ceramide may increase the plasma membrane content of ABCA1, leading to increased apoA-I binding and cholesterol efflux.

The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1.

Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport. However, the specifics of this interaction are unknown. It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter. Alternatively, apoA-I may bind directly to ABCA1 itself. To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux. We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP. Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type. We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10. However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus. From these observations, we propose an alternative model for apolipoprotein-mediated efflux.