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.
Highlights
Cholesterol is an essential constituent of all mammalian cell membranes and its availability is a prerequisite for cellular growth ly and other functions
N when murine erythroleukemia cell (MELC) were treated with hexamethylene bisacetamide (HMBA), o the inhibition of DNA synthesis caused by N HMBA treatment, was associated with a mulation of CE in tumoral cells, and a corresponding decrease of HDL cholesterol in the plasma compartment
Approximately the same extent as the acylCoA: cholesterol acyltransferase (ACAT) one of the mechanisms that cells use to control inhibitor, SaH. These results indicate the amount of toxic free cholesterol (FC); for the first time a role of cholesterol esterifica- under conditions of excess cholesterol, this is tion and of some related genes in differentia- transported from the plasma membrane to the Materials and Methods tion of erythroid cells
Summary
Data were reported as the average values±standard error (SE). Student’s t-test, suitably computed by means of the grouped. Protein content was e determined by Lowry et al.[29] us Cholesterol biosynthesis and esterification l To assess cholesterol biosynthesis, cells ia were incubated in medium containing 10% of c FBS with (14C)acetate (final radioactivity of 7.4 r MBq/mL) for 4 hours at 37°C in a CO2 incubae tor. Cells were incubated for 4 h at 37°C with (14C)oleic m acid (specific activity of 2.22 GBq/mmol, final radioactivity of 0.185 MBq/mL; Amersham m Biosciences) complex with BSA (essentially o fatty acid-free; Sigma). Relationship was linear for the cycle number used over the range 500-1500 ng of total RNA. Working in these conditions PCR products separated on agarose and stained with ethidium bromide were characterized by a major band of the predicted size (data not shown)
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