Cholesterol turnover in acute myelogenous leukemia with special emphasis on regulation of low density lipoprotein receptor expression in leukemic cells

Abstract

Cellular cholesterol requirements are met by receptor mediated uptake of cholesterol in low density lipoprotein (LDL) particles and/or by endogenous synthesis, the rate limiting enzyme in the synthesis pathway being 3-hydroxy-3-methylglutary coenzyme A (HMG-CoA) reductase. Expressions of both the LDL receptor and HMG-CoA reductase genes are normally under end product repression by cellular cholesterol. Leukemic cells from patients with acute myelogenous leukemia (AML) have a higher receptor mediated uptake of LDL than normal white blood and nucleated bone marrow cells and the levels of plasma cholesterol are decreased in AML patients. Previous studies have also found that LDL is a promising drug carrier candidate for cytotoxic agents. Expression of the drug transporter protein P-glycoprotein (Pgp) causes the multidrug resistance phenotype and recent studies suggest that Pgp may be involved in cholesterol transport.

The aims of the studies were to investigate the mechanisms behind elevated LDL receptor activity and hypocholesterolemia in AML and to investigate LDL receptor and HMG-CoA reductase activities in drug resistant leukemic cell lines.

In comparison with mononuclear blood cells from healthy individuals, cells from AM patients had elevated LDL receptor and HMG-CoA reductase activities as well as elevated RNA levels for both genes. LDL receptor RNA levels correlated weakly with protein activity which increased more than RNA levels during cholesterol deprivation suggesting post RNA regulation of the LDL receptor. AML cells showed a decreased sensitivity to the downregulatory effect of sterols on LDL receptor activity. The median sterol concentration for 50% inhibition (IC50) of LDL receptor activity was more than five times higher for AML cells than for normal cells and IC50 values correlated with the LDL receptor activity in freshly isolated AM cells (r = 0.53, P = 0.007) indicating that the elevated receptor activity may be a result of decreased sterol sensitivity. In spite of a higher growth rate, normal human fibroblasts, at low cell densities, were more sensitive to downregulation of LDL receptor activity by sterols and had a lower LDL receptor activity than subconfluent cells demonstrating that growth modulation of sterol sensitivity takes place in normal cells.

When mononuclear blood cells from healthy subjects were incubated in AML cell conditioned medium LDL receptor activity was induced by up to 240% and sensitivity to sterols decreased compared with cells incubated in control medium. LDL receptor activity in freshly isolated AML cells correlated with stimulation of LDL receptor activity in normal cells (r-0.79, P= 0.004). AML cells were also stimulated by their own conditioned medium. Interleukin-2 (IL-2) and IL-4 stimulated LDL receptor activity by up to 150% and caused a decreased sensitivity to sterols in normal cells. The stimulatory effect of AML cell conditioned medium on normal cells was not reversed by neutralizing IL-2, IL-4, IL6, or oncostatin M antibodies. Levels of 7alpha-hydroxy-4-cholesten-3- one, reflecting hepatic bile acid production, were markedly reduced in plasma from AML patients as compared with healthy subjects while 7-dehydrocholesterol levels, reflecting hepatic cholesterol synthesis, were similar. We found that 5 out of 5 drug resistant K562 cell lines had 2-10 times higher LDL uptake than the parental K562 cells. Drug resistant HL60 cell lines also had higher uptake of LDL than the parental cell line. Elevated uptake of LDL by drug resistant leukemic cell lines was not associated with an increased Pgp expression.

It is concluded that AML cells with elevated LDL uptake have a decreased sensitivity to the downregulatory effect of sterols on LDL receptor expression and that AML cells secrete a factor, possibly a growth factor, which stimulates LDL receptor expression. A paracrine or autocrine stimulation by such a factor could be responsible for the elevated LDL uptake in AML cells. AML patients have a reduced production of bile acids which could lead to cholesterol malabsorbition and hypocholesterolemia and several drug resistant leukemic cell lines had an elevated uptake of LDL which supports the idea of using LDL as a drug carrier.