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.

Probing the 121-136 domain of lecithin:cholesterol acyltransferase using antibodies.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121-136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121-136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121-136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121-136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121-136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121-136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.

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.

Dietary fat modulates serum paraoxonase 1 activity in rats.

We examined the effects of dietary fats with specific fatty acid compositions, on serum paraoxonase (PON1) activity in rats. Male adult Sprague-Dawley rats were divided randomly into four dietary groups. One group received the control diet [AIN 93M with soybean oil (5 g/100 g diet)], whereas the remaining three groups received the modified control diet supplemented with (15 g/100 g diet) triolein, tripalmitin or fish oil, respectively. After 20 d, blood was obtained after overnight food deprivation and PON1 activity was determined. Serum lipids and lipid components of lipoproteins were also determined. Serum PON1 activity [micromol/(L.min)] was significantly (P: < 0.05) higher in triolein (98 +/- 6) and lower in fish oil (41 +/- 4), compared with tripalmitin-fed rats (63 +/- 11). Serum PON1 activity in tripalmitin-fed rats was comparable to that of controls (67 +/- 9). Serum PON1 activity correlated significantly with serum lecithin:cholesterol acyltransferase (LCAT) activity (r = 0.77, P: < 0.001) and was transported in blood principally in association with the denser subfraction of HDL, very high density lipoprotein (VHDL; d > 1.15 kg/L). Serum PON1 activity correlated strongly with serum lipids as well as lipids of VLDL, HDL and its subfractions. Multiple linear regression analysis, however, showed a significant relationship of serum PON1 activity, principally with the phospholipids of VHDL (r = 0.47, P: < 0.002). These data suggest that the modulation of serum PON1 activity by dietary fat may be mediated via the effect of the specific fatty acids on the synthesis and secretion of VHDL, the subfraction of HDL that transports the majority of PON1 in the blood.

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.

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.

Evaluation of serum-lipid-related cardiovascular risk factors in Alzheimer's disease.

There is now sufficient evidence to suggest that cardiovascular pathology and altered lipid metabolism contribute to the development of late-onset Alzheimer's disease (AD). In the present study, 24 AD patients and 15 controls were assessed for cardiovascular risk based on serum lipid and lipid oxidation parameters. The AD patients appeared to have a more favorable cardiovascular risk profile than the controls based on high-density lipoprotein cholesterol (HDL-C) values. The levels of thiobarbituric-acid-reactive substances and the activity of the enzyme paraoxonase (PON) following copper oxidation indicate that female patients may have better protection against serum and perhaps tissue oxidants than males with AD. While the higher HDL-C values indicate lower cardiovascular risk, additional data on oxidized lipid parameters suggest a lower level of protection against serum oxidants in male AD probands. CopyrightCopyright 1999S.KargerAG,Basel

Decrease in high density lipoprotein cholesterol (HDL-C) levels following gemfibrozil therapy.

This report represents the continuation of our studies on the effects of gemfibrozil therapy on high density lipoprotein cholesterol levels. Previously, we reported that despite an impressive mean increase in high density lipoprotein cholesterol (20%), the response to 12 weeks of gemfibrozil therapy was highly variable. Accordingly, out of the 27 subjects studied, five actually had lower high density lipoprotein cholesterol at the conclusion of therapy compared to baseline values. The changes observed in plasma lipids, combined with correlational relationships suggest that the conversion of triglyceride rich lipoprotein components into high density lipoprotein may be impaired in those subjects that respond poorly or negatively to gemfibrozil therapy.

Fatty acids modulate lecithin:cholesterol acyltransferase secretion independently of effects on triglyceride secretion in primary rat hepatocytes.

The regulation of plasma lecithin:cholesterol acyltransferase (LCAT) expression is not well understood. Although oleic acid increases both the secretion of triglycerides and LCAT by primary rat hepatocytes, the effect of other fatty acids (FA) on LCAT secretion is not known. This study was designed to examine the effect of FA on the hepatic secretion of LCAT, triglyceride and apolipoprotein A-1 (apoA-1). Primary rat hepatocytes were incubated with serum-free medium, supplemented with individual FA (0-1 mmol/L) for 22-24 h. Preliminary studies indicated a linear secretion of LCAT up to 24 h in both control and FA-treated cells. When hepatocytes were incubated with 1 mmol/L FA, the LCAT secretion increased 50-100% (P < 0.01) in the presence of the 18-carbon FA (stearic, oleic, elaidic and linoleic acids), whereas the presence of butyric, lauric and palmitic acids had no significant effect. LCAT secretion decreased (P < 0.01) in the presence of docosahexaenoic acid (DHA). All FA (except DHA) significantly enhanced triglyceride secretion; however, only the 18 carbon FA significantly stimulated the synthesis and secretion of apoA-1 and secretion of LCAT. The secretion of LCAT correlated with apoA-1 secretion (r = 0.88, P = 0.004) but not with triglyceride secretion (r = 0.55, P = 0.12). Treatment with oleic acid resulted in a 1.5-fold increase in hepatocyte LCAT mRNA accumulation, whereas butyrate and palmitate had no effect. These data indicate that FA that promote the apparent synthesis and secretion of apoA-1 also stimulate the secretion of LCAT in vitro, suggesting a coordinate regulatory mechanism for apoA-1 and LCAT expression.

Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties.

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.

Purification of recombinant lecithin: cholesterol acyltransferase.

Production and purification of recombinant human lecithin:cholesterol acyltransferase (LCAT), secreted by baby hamster kidney (BHK) cells, has been improved by limiting the harvesting times for the conditioned medium and introducing an additional purification step. The recombinant BHK cells were grown until nearly confluent on multilayered flasks in a fetal-calf-serum-enriched medium. Subsequently, the cells were washed and supplied with serum free medium for 24-h periods. The conditioned medium, containing recombinant LCAT, was harvested at 24 and 48 h and subjected to chromatography on phenyl-Sepharose and ACA-44 agarose to isolate the recombinant enzyme. The second chromatography step revealed the presence of a low-molecular-weight contaminant that exhibited a carbohydrate/protein composition similar to proteoglycans. The major purified component contained LCAT activity and was homogeneous by acrylamide gel electrophoresis.

Evaluation of gemfibrozil therapy: predictive response from lipoprotein subfraction analysis.

Subjects with high-density lipoprotein cholesterol (HDL-C) values of less than 47 mg/dL (mean 35.6 +/- 5.5 mg/dL) were selected for this study to examine relationships between plasma lipids, lipoprotein components, and the outcome of gemfibrozil therapy. Changes in plasma lipoprotein subfractions were determined to better understand the previously observed variability of the responses in both HDL-C and triglycerides to gemfibrozil. Based on the data collected, an attempt was made to identify pretreatment lipid parameters that may be predictive regarding the efficacy of gemfibrozil therapy. Serum samples were analyzed at the outset and after the conclusion of 12 weeks of gemfibrozil therapy. Because the HDL-C response to therapy was highly variable, the data from patients were separated into two groups, responders (>20% increase in HDL-C) and nonresponders (<20% increase in HDL-C). The lipid components of lipoprotein subfractions were evaluated using multiple regression analysis yielding predictive models that show the relationship between specific lipoprotein subfractions and the percentage change in HDL-C and posttreatment triglyceride levels. Group classification was then predicted with 78% accuracy using specific lipoprotein subfractions to estimate an individual's percentage change in HDL-C. The major difference between the responder and nonresponder groups was their respective correlations between triglyceride-lowering and changes in HDL-C. In the responder group, there was a significant correlation between the changes in HDL-C and the lowering of triglycerides (r = 0.61, p = 0.03), whereas the nonresponder group showed no such correlation (r = 0.17, p = 0.52). The predictive model also proved to be highly accurate in forecasting the effectiveness of the triglyceride-lowering action of gemfibrozil in this group of patients.

Effect of interfacial pressure on the binding and phospholipase A2 activity of recombinant human lecithin-cholesterol acyltransferase.

We examined the effect of surface pressure on the interfacial binding and phospholipase A2 activity of lecithin-cholesterol acyltransferase. The enzyme bound to phosphatidylcholine monolayers with an apparent dissociation constant of 1.5 nM was excluded from the interface at pressures > 29 mN/m and exhibited maximal phospholipase activity at pressures between 29-28 mN/m. These data suggest that lipoprotein surface pressure may regulate lecithin-cholesterol acyltransferase activity in vivo.

The effect of captopril on the oxidation of plasma lipoproteins.

The antihypertensive drug captopril was found to inhibit the oxidation of low density lipoproteins by copper in a dose dependent manner in vitro. Up to 65% inhibition of oxidation was observed at the concentration of 100 micrograms/ml of captopril. During subsequent studies with patients, captopril protected low density lipoproteins against oxidation slightly better than enalapril, although this difference was not statistically significant. Captopril had no effect on the levels of Lp(a) as compared to the levels established during enalapril treatment.

2nd International Symposium on Reverse Cholesterol Transport: report on a meeting.

The purpose of this symposium was to provide a forum for presenting recent findings and for exchange of ideas concerning reverse cholesterol transport. Because the mechanism involving HDL in the prevention and/or reversal of atherosclerosis is still unknown, this area is of intense interest. Although reverse cholesterol transport as an essential physiological mechanism has been validated, the clinical significance of this pathway remains unclear. Key elements of knowledge are lacking that would allow the linking of cholesterol efflux from cells and tissues with specific events in HDL metabolism, particularly those that are relevant to the prevention and/or reversal of atherosclerosis. Because of the intricate nature of the interaction between the components of reverse cholesterol transport, this second conference involving the leading investigators proved particularly valuable for presentation and extensive discussion of unpublished and occasionally controversial findings. Following this conference, an agreement was reached between the organizers and Dr. Norman E. Miller to combine this symposium with the previously held International Symposia on High Density Lipoproteins in Atherosclerosis (organized by Dr. Miller).

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.

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.

Expression and characterization of recombinant human lecithin:cholesterol acyltransferase.

We have established a baby hamster kidney (BHK) cell line that constitutively expresses significant quantities of human recombinant lecithin:cholesterol acyltransferase (rLCAT). LCAT cDNA was cloned into a mammalian expression vector containing the metallothionein promoter and the dihydrofolate reductase gene. After transfection, the BHK cells were treated with 500 microM methotrexate for 2 weeks to select the successfully transfected cells. Surviving colonies were subcloned and high level secretors were identified by measurement of LCAT activity and mass in the culture medium. The attachment of transfected cells to microcarrier beads enabled the efficient production of large quantities of rLCAT in a serum-free medium. After a single-step chromatography procedure, the rLCAT was purified to homogeneity with yields exceeding 1 mg of rLCAT per 100 ml of culture medium. The molecular weight of rLCAT (approximately 66,000) was identical to that of purified human plasma LCAT on SDS polyacrylamide electrophoresis. The rLCAT was activated by apolipoprotein A-I and had an average specific activity that was similar to purified plasma LCAT. After selective deglycosylation with either neuraminidase or N-glycanase, rLCAT and plasma LCAT had identical molecular weights. The simplification of the production and purification of rLCAT reported here will enable a more in depth analysis of the structure and function of this enzyme.