Doxorubicin transport by RALBP1 and ABCG2 in lung and breast cancer.

RALBP1 (RLIP76) is the major transporter of doxorubicin (DOX) in lung cancer cells, and that the difference in sensitivity of small cell lung cancer (SCLC) cells to DOX is due to differential phosphorylation by PKCalpha. Our recent studies have suggested that RALBP1 present in MCF-7 breast cancer cells has significantly lower specific activity for transport of DOX than wild-type recombinant protein, and its level of expression is significantly lower than that in lung cancer cells. In the present study, we have explored whether or not this is a generalized phenomenon for breast cancer, and have compared the relative contributions of RALBP1 and the ABC-family transporter, ABCG2 to total DOX transport activities in two SCLC (H1417 and H1618), two non-small cell lung cancer (NSCLC) (H358 and H520), and three breast cancer (T-47D, MDA-MB231, and MCF-7) cell lines. Results of these studies show lower protein expression and specific activity of RALBP1 in all three breast cancer cell lines as compared with lung cancer cell lines. Furthermore, we demonstrate that RALBP1 contributes only a minor fraction of DOX transport activity in breast cancer cell lines, suggesting that greater DOX sensitivity of breast cancer may be related to lower RALBP1 transporter activity and that the transport mechanisms involved in multidrug resistance of lung and breast cancer are distinct.

Isolation and characterization of lecithin-cholesterol acyltransferase from hog plasma.

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified from hog plasma by a highly efficient procedure. The final enzyme preparation was purified 30,000-fold over the starting material and was homogeneous as indicated by polyacrylamide gel electrophoreses in the presence of both SDS and urea. The purified hog lecithin-cholesterol acyltransferase had an apparent molecular weight of 66 000 on SDS-polyacrylamide gel electrophoresis and HPLC and was found to contain about 21.4% (w/w) carbohydrate-hexose, 11.3%; hexosamine, 1.9%; sialic acid, 8.2%. The amino acid composition analysis showed that hog lecithin-cholesterol acyltransferase contains four half cystines per mol; two cysteines were titrated at neutral pH with 5,5'-dithiobis(2-nitrobenzoic acid). Nearly all the phenolic groups were unavailable to the solvent at neutral pH, while they become exposed at around pH 11. Hog lecithin-cholesterol acyltransferase was found to be associated with HDL in the plasma and it prefers HDL as a substrate. The physicochemical properties of hog lecithin-cholesterol acyltransferase were generally similar to those of the human and the rat enzyme.

Study of the lecithin: cholesterol acyltransferase reaction with liposome and high density lipoprotein substrates.

The activity of highly purified preparations of human plasma lecithin: cholesterol acyltransferase were stabilized by precipitating the enzyme with ammonium sulfate and using the dilutions of the particulate lecithin: cholesterol acyltransferase suspension for enzyme assays. Ammonium sulfate concentrations in the assay mix up to 0.1 M had no significant effect on lecithin: cholesterol acyltransferase activity. The basic enzymatic properties of lecithin: cholesterol acyltransferase were investigated using liposomes and high density lipoprotein (HDL) substrates. pH optima for both substrates was approximately 8.0. The temperature dependence of lecithin: cholesterol acyltransferase activity resulted in non-linear Arrhenius plots with both substrates. The activity vs. temperature (degrees C) curves showed slight inflections at 30 degrees C, which may have been due to the relatively rapid inactivation of the enzyme above this temperature. HDL3 was found to be a better substrate than HDL or HDL2. HDL3 was also considerably better than egg phosphatidylcholine/cholesterol liposomes as an lecithin: cholesterol acyltransferase substrate. Addition of HDL2 to a reaction mix of enzyme and HDL3 indicated that HDL2 acts as an inhibitor of cholesterol esterification in this system.

Lecithin:cholesterol acyltransferase in familial HDL deficiency (Tangier disease).

These studies were performed to investigate the relationship between the enzyme lecithin:cholesterol acyltransferase and plasma lipoproteins in Tangier disease, a condition characterized by a virtual absence of high-density lipoproteins (HDLs) and an accumulation of cholesteryl esters in peripheral tissues. Apolipoprotein A-I was nearly absent from the patient's plasma (1% of the normal levels were found). However, apolipoprotein A-I purified from the plasma of the Tangier disease patient, was found to activate both purified and the plasma enzyme. At lower apolipoprotein concentrations (up to 25 micrograms/ml), about twice the amount of Tangier apolipoprotein A-I was required to achieve a certain level of lecithin:cholesterol acyltransferase activity as compared with the activating potential of the normal apolipoprotein. Gel chromatography studies revealed that as in normal plasma, lecithin:cholesterol acyltransferase in Tangier plasma was associated with an HDL-size lipoprotein fraction. However, unlike in normal plasma, this lipoprotein complex (containing lecithin:cholesterol acyltransferase) was not removed from Tangier plasma by immunoaffinity chromatography utilizing immobilized anti-apolipoprotein A-I antibodies. Plasma incubation studies showed that free cholesterol was primarily supplied by LDL in normal plasma, whereas both LDL and VLDL donated the free cholesterol for lecithin:cholesterol acyltransferase reaction in Tangier plasma. The majority of the cholesteryl esters, generated during the incubation experiments, were transferred back to LDL in normal plasma, whereas in Tangier plasma both LDL and VLDL served as cholesteryl ester acceptors. The cholesteryl ester transfer from HDL to lower-density lipoproteins was lower in Tangier plasma as compared to this process in a normal control, suggesting that a minimal cholesteryl ester core may be required for the stability of HDL.

Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source.

Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.

The metabolism of high-density lipoproteins.

Although high-density lipoproteins (HDLs) have been shown to be the best single indicator of the risk of coronary heart disease (CHD), relatively little is known about their metabolism. Accordingly, only limited strategies are available for therapeutically raising plasma HDL levels. The circulating HDL particle is assembled in the blood as the result of remodeling the nascent discoidal HDL followed by transfer of lipid and protein components from other lipoproteins. The catabolism of HDL is equally complex. The receptor-mediated removal mechanism of HDL from the plasma has yet to be substantiated. Despite the extensive studies performed, no clear mechanism has emerged whereby HDL particles protect the arteries from atherosclerosis. Reverse cholesterol transport remains an attractive hypothesis, but several other potential mechanisms may also play a role in the interaction between HDL and the arterial surface. Recent studies related to the regulation of HDL metabolism are discussed with particular emphasis on the potential role of the postprandial state. A brief discussion is also provided on potential future strategies for regulating HDL levels through pharmacologic intervention.

Effect of ethinyl estradiol treatment on lipoproteins and LCAT activity in aged rats.

The induction of hepatic lipoprotein (apo B/E) have been investigated in Fischer-344 rats. These studies were aimed to determine the mechanism underlying the previously observed (Lee et al., Mech. Ageing Dev., 61 (1991) 85-98) hypercholesterolemia and the age-related decrease in the fractional rate of endogenous cholesterol esterification. Young (5 months) and aged (22 months) male Fischer-344 rats were treated with pharmacological doses (5 mg/kg per day) of ethinyl estradiol (EE) for 7 days. Reduction of plasma cholesterol (57% in young vs 47% in aged rats) and high density lipoprotein cholesterol (64% in young vs 63% in aged rats) occurred in both groups upon EE treatment. Initial low density lipoprotein levels were very low in the plasma of young rats and consequently were not affected by EE treatment. However, in aged rats, the low density lipoprotein levels were much higher initially and were markedly reduced by EE treatment. (18.0 vs 10.0 mg/dl). Very low density lipoproteins were about the same initially but increased in aged rats and decreased in young rats upon EE treatment. Both the lecithin:cholesterol acyltransferase (LCAT) activity (as determined with a proteoliposome substrate) and the fractional rate (FR) of the endogenous cholesterol esterification decreased in treated animals compared to controls. However, the differences in the FR of the endogenous cholesterol esterification between young and aged rats (observed before treatment) were nearly abolished upon treatment. These data suggest that the previously observed age related decrease in the FR of endogenous cholesterol esterification is due to the accumulation of apolipoprotein E-rich (apo E) lipoproteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Age related changes in the lipoprotein substrates for the esterification of plasma cholesterol in rats.

The activity of the enzyme lecithin:cholesterol acyltransferase (LCAT) and the properties of its lipoprotein substrates have been investigated in 6- and 19-month-old Fischer-344 rats. These studies were carried out to determine the nature of the relationship between the observed hypercholesterolemia and the age-related decrease in the fractional rate of lipoprotein cholesterol esterification. The distribution of LCAT activity of plasma fractions was determined following gel chromatography and ultracentrifugation respectively. LCAT activity was found to be associated with the high density lipoprotein (HDL) fraction when rat plasma was passed through a Bio-Gel A-5 M column. Upon density gradient ultracentrifugation for 24 h it was found associated with HDL fraction; d = 1.125-1.21 g/ml. However, following prolonged ultracentrifugation (40 h), the majority of the LCAT activity was displaced into the lipoprotein-free infranatant (d greater than 1.225 g/ml). The dissociation of LCAT from its complex with HDL occurred to a smaller extent in aged rat plasma than in young rat plasma. Substrate specificity studies indicated that HDL was a considerably better substrate for LCAT than very low density lipoproteins (VLDL) in both young and aged rats. In addition, HDL from young rats was a better substrate for LCAT than the HDL from aged rats. Incubation experiments followed by the isolation of lipoproteins and the subsequent analyses of their cholesterol contents revealed that the age-related hypercholesterolemia was mainly due to an increase in the cholesterol carried by lipoprotein fractions d = 1.025 -1.07 g/ml (LDL + HDL1). These and other low density lipoproteins (d less than 1.025 g/ml) were poor substrates for LCAT. However, these lipoproteins could provide free cholesterol for esterification by first transferring it to HDL (d = 1.07-1.21). The HDL isolated from the plasma of aged rats was enriched with apolipoprotein (apo) E and these lipoprotein particles were found to be inferior substrates for LCAT. These data suggest that the decreased fractional rate of esterification observed in aged rats is due to the slower utilization of the HDL lipid substrate pool by the enzyme LCAT as a result of the accumulation of unfavorable substrates (compositionally altered HDL particles) for the LCAT reaction.

Age-related changes in the rate of esterification of plasma cholesterol in Fischer-344 rats.

Plasma cholesterol and triglyceride levels and selected molecular species of plasma cholesteryl esters and triglycerides were determined in 6-, 12-, 15-, 18-, 21-, and 24-month-old Fischer-344 rats. Lecithin:cholesterol acyltransferase (LCAT) activity was also determined using two independent methods utilizing endogenous and exogenous substrates. Plasma cholesterol levels increased up to 18 months of age and then plateaued. Of the plasma triglyceride molecular species investigated (C50, C52, C54 and C56), only the levels of C52 increased linearly with age. The concentration of other triglyceride molecular species did not change with age. The fractional rate of plasma cholesterol esterification showed a decreasing trend with age, whereas, the net cholesterol esterification rate showed a gradual age related increase. However, this latter parameter remained unchanged with age when the data were normalized for body weight. The cholesterol esterification rates measured using an exogenous substrate (estimating LCAT enzyme levels) showed essentially no change with age. These data indicate that changes in the levels and/or composition of lipoprotein substrate(s) for LCAT are likely causes of the observed age-related changes in the fractional rate of plasma cholesterol esterification. The net esterification rate of plasma cholesterol was significantly correlated with the plasma triglyceride levels when the animals for all age groups were treated as one experimental group.

Plasma lipids and cholesterol esterification in Alzheimer's disease.

Eight patients and eight age matched controls were recruited to study parameters related to plasma lipoprotein metabolism in Alzheimer's disease based on previous studies in Down's syndrome (A.G. Lacko et al., Clin. Chim. Acta, 132 (1983) 133). The fractional rate of cholesterol esterification (% cholesterol esterified per hour) was 16% lower in the patient group compared with controls. Correlational analyses of lecithin/cholesterol acyltransferase (LCAT) activity and plasma lipids revealed additional differences between the Alzheimer's patients and control subjects. These data are strikingly similar to those obtained earlier with Down's syndrome patients. These data, combined with analyses of cholesteryl ester transfer protein (CETP) levels, suggest that reverse cholesterol transport in general and CETP activity in particular may be altered in Alzheimer's disease.

Relationship between cholesteryl ester transfer activity and high density lipoprotein composition in hyperlipidemic patients.

Cholesteryl ester transfer from solid-phase bound HDL to endogenous plasma HDL or VLDL/LDL was determined in 50 patients with primary disorders of lipid metabolism and 27 normolipidemic subjects. Transfer to the plasma HDL pool was significantly reduced in familial hypercholesterolemia, familial combined hyperlipidemia, hypoalphalipoproteinemia and dysbetalipoproteinemia. Subfractionation of HDL revealed that the lipid transfer to HDL3 was significantly reduced in all patient groups while transfer to HDL2 was increased in those with dysbetalipoproteinemia and familial hypertriglyceridemia. Transfer to LDL and VLDL was increased only in patients with dysbetalipoproteinemia and hypoalphalipoproteinemia. Reduced transfer to HDL occurred in samples with altered HDL composition; particularly where HDL-triglyceride was significantly increased and HDL-cholesteryl esters were reduced. Transfer of cholesteryl ester to HDL3 was significantly decreased in patients with vascular disease. These findings indicate that impaired interaction of cholesteryl ester transfer protein with the HDL3 pool may contribute to the risk of coronary heart disease in patients with specific plasma lipid abnormalities.

On the interpretation and potential diagnostic value of the measurements related to lecithin:cholesterol acyltransferase activity.

At least one reliable method is available for measuring LCAT activity in human subjects(25), and this assay may help to probe even subtle changes in plasma lipids and lipoproteins. Since plasma lipids and lipoproteins are known to be present in abnormal amounts in a large number of metabolic diseases, the method of measuring the rate of cholesterol esterification in plasma could assume a substantial role in the diagnosis and (or) monitoring of treatment of pathologic states.

Lecithin: cholesterol acyltransferase in Down's syndrome.

Based on earlier reports indicating that Down's syndrome may represent an atheroma-free human model, two groups of institutionalized subjects were compared with respect to various parameters of their plasma lipid transport system. One group of subjects was comprised of Down's syndrome subjects and the second, a group of mentally retarded individuals. Parameters measured included plasma cholesterol, triglyceride, HDL-cholesterol, apolipoprotein levels (A-I, B, C-III, and E), lecithin:cholesterol acyltransferase (LCAT) activity, body mass and blood pressure. Statistical analyses indicated no significant differences between the two groups except for the lower fractional rate of cholesterol esterification (% cholesterol esterified per hour, p = 0.0049) in the Down's syndrome subjects. Adjustment for the effects of body mass and age revealed no other significant differences between the two groups except for a lower molar rate of esterification (nmol cholesterol esterified X h-1 X ml-1, p less than 0.0063) in the Down's syndrome subjects. Additional differences between the two groups were revealed by partial correlational analyses of LCAT activity with the measured parameters or ratios of these parameters which suggests that the composition and/or metabolism of lipoproteins may differ between these two groups. Whether the lower LCAT activity and the other differences reflected by the correlational analyses contribute to the decreased incidence of atherosclerotic lesions in Down's syndrome remains to be elucidated.

High cholesterol diet down regulates the activity of activator protein-1 but not nuclear factor-kappa B in rabbit brain.

Cardiovascular risk factors and alterations in cholesterol metabolism are implicated in the pathogenesis of Alzheimer's dementia (AD). The hypercholesterolemic rabbit model of atheroslerosis and AD was utilized in this study to examine oxidative stress related changes in the brain. The high cholesterol diet induced dramatic increases in plasma and liver cholesterol concentrations, but brain cholesterol levels remained constant. Similar effects have been found regarding lipid oxidation products. The amounts of conjugated dienes, trienes and thiobarbituric acid reactive substances (TBARS) significantly increased in the plasma of cholesterol treated animals while the brain cortex showed no signs of increased lipid peroxidation. The oxidative damage sensitive nuclear transcription factor kappa B (NF-kappaB) and activator protein-1 (AP-1) diverged in their responses. Accordingly, the AP-1 DNA binding activity decreased by more than 50% in brain nuclear protein extracts while the NF-kappaB binding activity remained unaltered by the hypercholesterol diet. These results indicate that despite the relative resistance of the central nervous system to dietary manipulation of its lipid composition and lipid peroxidation products, chronic dietary intake of cholesterol can alter the function of certain proteins involved in regulation of gene expression in the brain.

Apolipoprotein D in the aging brain and in Alzheimer's dementia.

Apolipoprotein D (apoD) levels were examined in the temporal cortex as well as an assessment of the location of apoD positive cells within the brain by immunohistochemical and biochemical methods in young control (YC), aged control (AC), and Alzheimer's demented (AD) probands. Scattered apoD positive astrocytes and oligodendrocytes were found throughout the white matter by immunohistochemistry. ApoD immunoreactivity was also observed in the cerebellar oligodendrocytes of the YC group. There was faint positive apoD staining in scattered cortical astrocytes and a few neurons in the same group. In contrast, some of the AC and all of the AD probands had intense and frequent apoD immunostained cortical astrocytes and pyramidal neurons. The cortical senile plaques and neurofibrillary tangles were apoD immunonegative. No quantitative differences were found between the cortical apoD levels in the AC and AD groups, determined by immunoblotting. ApoD detected in the brain tissue was different in molecular weight (29 kDal) from that seen in CSF or in the serum (32 kDal). Our results indicate apoD is present in the human brain, especially in glial cells, and has increased abundance in the elderly and AD subjects.

Decreased lecithin:cholesterol acyltransferase activity in the plasma of hypercholesterolemic pigs.

Lecithin:cholesterol acyltransferase (LCAT) activity levels were determined, as function of plasma total cholesterol (TC) in 13 normocholesterolemic (TC less than 85 mg/dL) and in 28 hypercholesterolemic (TC greater than 98 mg/dL) pigs. The normocholesterolemic group consisted of pigs that carried apo-B allelic genes other than Lpb5 and or Lpb8. The hypercholesterolemic group consisted of Lpb5/x and Lpb5/8 heterozygous and Lpb5/5 homozygous animals. The data reported in this study show that the LCAT activity in the plasma of hypercholesterolemic (HC) pigs (79 +/- 43 units) was significantly lower (p less than 0.0005) compared to the normocholesterolemic controls (175 +/- 45 units). Furthermore, LCAT activity was positively correlated with TC in the normocholesterolemic group (r = +0.54; p less than 0.05), whereas it was negatively correlated with TC in the hypercholesterolemic group (r = -0.73; p less than 0.001). Additional data obtained from incubation experiments suggest that the lower LCAT activity in hypercholesterolemic pigs may be due, at least in part, to inhibition of LCAT activity by components found in the lipoprotein-deficient fractions of the plasma of hypercholesterolemic pigs.

Age-related changes in plasma lipid levels and tissue lipoprotein lipase activities of Fischer-344 rats.

Fischer-344 rats of 6, 12, 18, 21 and 24 mth of age were investigated. The data were collected from groups of rats ranging from 5-10 rats per age group. Modest age-related increases were noted in body weight, plasma triglycerides, total cholesterol and free (unesterified) cholesterol. Much more significant changes were noted in tissue lipoprotein lipase activities where the adipose tissue lipases declined sharply (r = -0.90, P less than 0.0001; r = -0.86, P less than 0.0001) and the muscle tissue lipases decreased moderately (r = -0.86, P less than 0.0001; r = -0.58, P less than 0.0001) with age. These observed trends suggest that enzymatic parameters, specifically tissue lipoprotein lipase activities, may be considerably more accurate indicators of age-related physiological changes than levels of plasma lipids such as cholesterol and triglyceride.

Physiologic role and clinical significance of reverse cholesterol transport.

Low levels of high-density lipoproteins have been consistently shown to be a major risk factor for coronary heart disease. However, the precise role of HDL in the prevention or reversal of atherosclerosis (or both) is unknown. It has been proposed that HDL functions jointly with the enzyme lecithin:cholesterol acyltransferase and the cholesteryl ester transfer protein to facilitate the movement of cholesterol from tissues to the liver. This mechanism--referred to as reverse cholesterol transport--has been shown to be an important physiologic mechanism. However, its clinical significance, though intriguing, is unclear. This article reviews recent advances concerning the components of reverse cholesterol transport and evaluates their potential significance in the early diagnosis and treatment of atherosclerosis.