Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells

Abstract

Cholesterol is an essential constituent of all mammalian cell membranes and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3) and acylCoA: cholesterol acyltransferase (ACAT) and cholesterol export (caveolin-1) in Friend virus-induced erythroleukemia cells (MELC), in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA). FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells.

Keywords: cholesterol; cholesterol esterification; differentiation.; erythroleukemia.

Figure 1

Time course of cell growth, ³H-thymidine incorporation and protein content in murine erythroleukemia cells (MELC) in presence or absence of hexamethylene bisacetamide (HMBA). MELC cells were seeded at a density of 1×10⁵ cells/mL and stimulated to growth by adding 10% FCS. At the indicated times A) cell number, B) ³H-thymidine incorporation and C) protein content were determined as described in Materials and Methods. Data are mean±Standard Error of three independent experiments, each performed in triplicate. *Indicates statistically significant (P<0.05) compared with MELC.

Figure 2

Time course of ¹⁴C-acetate incorporated into A) free cholesterol, B) colesterol esters and C) 14C-oleate incorporation into Colesterol Esters of murine erythroleukemia cells (MELC) in presence or absence of hexamethylene bisacetamide (HMBA). MELC cells were seeded at a density of 1×10⁵ cells/mL and stimulated to growth by adding 10% FCS ¹⁴C-acetate and ¹⁴C-oleate were added 3 h before each time point. MELC were harvested every 24 h for a total period of 96 h. Data are mean±Standard Error of three independent experiments, each performed in triplicate. *Indicates statistically significant (P<0.05) compared with MELC.

Figure 3. Multidrug Resistance (MDR-1), Acyl-coenzyme A: Cholesterol Acyl Transferase (ACAT-1) and Caveolin-1 mRNA levels in murine erythroleukemia cells (MELC) and hexamethylene bisacetamide (HMBA)-induced MELC. The figure shows representative autoradiograms of MDR-1, ACAT-1, caveolin-1 (Cav-1) and β-actin mRNA in MELC and in HMBA treated MELC. Reverse transcription-polymerase chain reaction analysis was performed as described in Materials and Methods.

Figure 4

Relative levels of Acyl-coenzyme A: Cholesterol AcylTransferase (ACAT-1), Multidrug Resistance (MDR-3) and Caveolin-1 (C) mRNA in murine erythroleukemia cells (MELC). Reverse transcription-polymerase chain reaction analysis was performed for three independent experiments. Relative levels of A) ACAT-1, B) MDR-3 and C) Caveolin-1 (Cav-1) mRNA were normalized to the corresponding β-actin levels and expressed as percentage.

Figure 5

Photographs of murine erythroleukemia cells (MELC) cultures with or without treatment with hexamethylene bisacetamide (HMBA) and acyl amide ACAT inhibitor (SaH). A) Microphotographs taken by using an inverted microscopy of untreated or HMBA and SaH treated- MELC 72 h after culture. B) Microphotograpfs of the above cell cultures examined by inverted microscopy stained with ORO for neutral lipids and counter-stained with hematoxylin for nuclei.