Cholesterol released from ingested low density lipoproteins in lysosomes moves both to the plasma membrane and to the endoplasmic reticulum (ER) where it is re-esterified. Whether cholesterol can move directly from lysosomes to ER or first must traverse the plasma membrane has not been established. To examine this question, the endocytic pathway of rat hepatoma cells was loaded at 18°C with low density lipoproteins (LDL) labeled with [ 3 H]cholesteryl linoleate, and the label then was chased at 37°C. The hydrolysis of the accumulated ester proceeded linearly for several hours. Almost all of the released [ 3 H]cholesterol moved to the plasma membrane rapidly and without a discernable lag. In contrast, the re-esterification in the ER of the released [ 3 H]cholesterol showed a characteristic lag of 0.5-1 h. These data are inconsistent with direct cholesterol transfer from lysosomes to ER; rather, they suggest movement through the plasma membrane.Furthermore, we found that progesterone, imipramine and 3-␤-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) strongly inhibited the re-esterification of lysosomal cholesterol in the ER. However, contrary to previous reports, they did not block transfer of [ 3 H]cholesterol from lysosomes to the cell surface. Therefore, the site of action of these agents was not at the lysosomes. We suggest instead that their known ability to block cholesterol movement from the plasma membrane to the ER accounts for the inhibition of lysosomal cholesterol esterification.These findings are consistent with the hypothesis that cholesterol released from lysosomes passes through the plasma membrane on its way to the ER rather than proceeding there directly. As a result, ingested cholesterol is subject to the same homeostatic regulation as the bulk of cell cholesterol, which is located in the plasma membrane.Cell cholesterol levels are tightly regulated by homeostatic mechanisms. For example, when cells ingest cholesterol in the form of low density lipoprotein (LDL), 1 sterol biosynthesis is reduced and excess cholesterol is converted to cholesteryl esters for storage (1, 2). While these processes are well understood, the associated pathways of intracellular cholesterol movement are obscure. It appears that cholesterol moves bidirectionally between the plasma membrane and the ER (3). Furthermore, the cholesterol released from ingested LDL in lysosomes moves rapidly to the plasma membrane (4). Since the cholesterol derived from the degradation of LDL is reesterified by ACAT (1, 2), lysosomal sterol must also be transported to the ER.Two recent studies addressed the question of the pathway taken by cholesterol from lysosomes to ER. One suggested direct movement of cholesterol from lysosomes to ER by a mechanism that is inhibited by amphiphiles (5). The other concluded that approximately 70% of lysosomal cholesterol passes through the plasma membrane prior to esterification, the remainder moving directly between the lysosomes and the ER (6).In the present study, we have analyzed the fate of inge...

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The silencing of tumor suppressor genes associated with increased DNA methylation of the promoter regions is a frequent observation in many forms of cancer. Reactivation of these genes using pharmacological inhibitors of DNA methyltransferase such as 5-aza-29-deoxycytidine (decitabine) is a worthwhile therapeutic goal. The effectiveness and tolerability of low-dose intravenous and subcutaneous decitabine regimens to demethylate and reactivate expression of the methylated c-globin gene in baboons and in patients with sickle cell disease led to successful trials of low-dose regimens of this drug in patients with myelodysplastic syndrome. Since these low-dose regimens are well-tolerated with minimal toxicity, they are suitable for chronic dosing to maintain promoter hypomethylation and expression of target genes. The development of an orally administered therapy using DNA methyltransferase inhibitors would facilitate such chronic approaches to therapy. We tested the ability of decitabine and a new salt derivative, decitabine mesylate, to reactivate the methylated c-globin gene in baboons when administered orally. Our results demonstrate that oral administration of these drugs at doses 17-34 times optimal subcutaneous doses of decitabine reactivates fetal hemoglobin, demethylates the e e-and c-globin gene promoters, and increases histone acetylation of these promoters in baboons (Papio anubis). Am. J. Hematol. 82:981-985, 2007.