Synthesis of nuclear lipids in L2C leukemic lymphocytes.

We previously reported that propiconazole strongly inhibits cholesterol synthesis, but not cell division in a stimulated cell, the human lymphocyte cultured with phytohemagglutinin, showing that newly synthesized cholesterol is not necessary for cell division. In this study we labeled the L2C leukemic guinea pig lymphocyte, a naturally stimulated cell, with [2-14C]acetate, and compared the composition of newly synthesized lipids isolated from nuclei and whole cells (or microsomes). We observed that the proportion of cholesterol in labeled non-saponifiable lipids extracted from nuclei was lower than in non-saponifiable lipids isolated from whole cells, whereas the proportion of squalene and polar lipids was higher. By analyzing total lipid extracts, the polar lipids were identified as alkylglycerols, and the above mentioned distribution of constituents was confirmed. The identification of alkylglycerols was also supported by the comparison of radioactive lipid composition after labeling cells with three different lipid precursors: [2-14C]mevalonate, [2-14C]acetate and [2-14C]stearate. When cells were labeled in the presence of dodecylimidazole, the percentage of squalene and alkylglycerols decreased in nuclear lipids, but was not altered when cells were cultured in the presence of propiconazole, a cholesterol synthesis inhibitor which does not affect cell division of human stimulated lymphocytes. We have shown that dodecylimidazole inhibited alkylglycerol biosynthesis and squalene uptake by the nucleus, suggesting that these compounds could play a role in the regulation of cell division.

Active cholesterol biosynthesis in cultured aortic smooth muscle cells: evolution during the life-span of the culture.

Cultured aortic smooth muscle cells from rabbit, in synthetic and contractile state, are considered good models for studying pathological and normal cells, respectively, during the atherosclerotic process. Cholesterogenic activity was compared in cells which were obtained in both states of the same subculture and incubated with labeled sodium acetate. This activity (expressed as the percentage of total cell radioactivity uptake transformed into cholesterol) was very high in synthetic cells and comparable to that of cancer cells. Cholesterol synthesis was lower in contractile cells and similar to that observed in a nonpathological cultured cell. During the cell life-span (studied in two cultures) cholesterogenic activity initially increased and then slowly decreased, in the two phenotypic states. Near the end of the culture life, cholesterol production drastically decreased, but this was due to a blocking of the last steps, lanosterol demethylation and C27 sterol transformation into cholesterol, rather than to a sharp decrease in the first steps of the cholesterogenic process. Cells in the synthetic and contractile states released newly synthesized lipids which were essentially late precursors of cholesterol, but accumulation of oxy-sterols was not observed. The excretion of metabolites increased with culture aging.

Metabolism of low-density lipoprotein in differentiated and undifferentiated HT29 colon cancer cells.

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.

Cholesterogenesis and cell division in phytohemagglutinin-stimulated human lymphocytes: a comparative study with several inhibitors.

It has been shown that when lymphocytes are stimulated by phytohemagglutinin the expected stimulation of DNA synthesis is preceded by stimulation of cholesterol synthesis. This confirms the existence of a relation between cell division and cholesterol synthesis. We studied the effect on cell division of six inhibitors of cholesterol biosynthesis, previously shown to interfere with different steps of the process: 7 beta-hydroxycholesterol, 25-hydroxycholesterol, lanost-7-en-3 beta, 32-diol, mevinolin, propiconazole, dodecylimidazole. Since experiments were performed in the presence of a high percentage of human serum, which provided cells with exogenous cholesterol via the LDL-receptor pathway, our investigation was focused on the role of newly synthesized cholesterol. The biosynthesis was evaluated by labeling cells with [14C]sodium acetate; to take into account variations of cell permeability to sodium acetate, the results were expressed as the percentage of total cellularly incorporated radioactivity transformed into cholesterol, after separation from all other labeled metabolites. These data were compared with the percentage of transformation into nonsaponifiable lipids, which varied in parallel with HMG-CoA reductase activity, as confirmed by direct enzymatic measurement. Cell division was assessed by simultaneous measurements of three parameters: thymidine incorporation into DNA, cell proliferation and cellular protein content. All the effectors strongly inhibited the conversion of labeled acetate into cholesterol, but cell division was not inhibited by two of them: propiconazole and 7 beta-hydroxycholesterol. These compounds only slightly inhibited the synthesis of nonsaponifiable lipids, which mainly consisted of methylsterols resulting from a blockage of lanosterol demethylation. Thus, it can be concluded that the nonsaponifiable metabolite essential for cell growth is not newly synthesized cholesterol. It was also found that inhibitors affected cell division only when they were added to the culture medium before the decline of cholesterol synthesis stimulation.

Effect of estradiol and antiestrogens on cholesterol biosynthesis in hormone-dependent and -independent breast cancer cell lines.

The effects of estradiol and/or antiestrogens on cholesterol biosynthesis were studied in two breast cancer cell lines. Cholesterogenic activity was evaluated after labeling cells with sodium [14C]acetate for increasing periods of time (up to 24 h) and measuring the incorporation of the radioactivity into nonsaponifiable lipids and into cholesterol, after separation from other labeled metabolites. We compared the effects of estradiol on cholesterogenesis with the well-known effects of this hormone on cell proliferation: estradiol stimulated both cholesterol synthesis and cell growth in MCF-7 cells, but stimulated neither in BT20 cells. The stimulation affected both the 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase step and the post-HMGCoA steps. Only the key enzyme step appeared to be mediated by the estrogen receptor. The hydroxytamoxifen and LY 117018 antiestrogens strongly inhibited cellular cholesterol production in both cell lines. Under the same conditions, cell growth is affected in MCF-7 cells, but not in BT20 (as shown by groups from other laboratories). This demonstrates that de novo synthesis of cholesterol is not essential for cell growth when cells are cultured in the presence of whole serum. The inhibition of cholesterol synthesis by antiestrogens mainly affected the lanosterol demethylation step and the C-27 sterol to cholesterol conversion. This inhibiting effect of antiestrogens was not mediated by the estrogen receptor.

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis.

The effects of estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10(-10) M E2 stimulated and 10(-8) M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E2. E2 was without effect in the HTC and estrogen-resistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tam-resistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E2 occurred via a different pathway that appears to be related to the presence of ER in the cells.

Regulation of cholesterol biosynthesis at a stage after the 3-hydroxy-3-methylglutaryl-CoA reductase step, in normal and leukemic (L2C) guinea pig lymphocytes.

Cholesterogenic activity in normal and leukemic guinea pig lymphocytes was measured by incorporation of labeled sodium acetate into cholesterol, after separation from other labeled metabolites. Our study is in agreement with the large difference previously found between the two kinds of cells at the 3-hydroxy-3-methylglutaryl-CoA reductase step, but it also shows that the difference is not as great as described earlier, when expressed in terms of the final product, cholesterol. This is mainly due to differences in the analytical methods. Our more detailed procedure showed a blockage of cholesterol synthesis in leukemic guinea pig lymphocytes (L2C cells) at the step of lathosterol (cholest-7-en-3 beta-ol) isomerization, and a higher plasma membrane permeability of these cells for sodium acetate, compared to normal cells. The lack of cholesterogenesis regulation by low density lipoproteins in L2C cells, previously reported after measuring 3-hydroxy-3-methylglutaryl-CoA reductase activity, was confirmed with regard to cholesterol itself, as well as the usual regulation of normal cells, which appeared to occur also at a post-hydroxymethylglutaryl-CoA step.

Inhibition, post-hydroxymethylglutaryl-CoA regulation and relation to cell growth of cholesterol biosynthesis in cultured human skin fibroblasts.

Cholesterol biosynthesis in cultured human skin fibroblasts was studied by [14C]acetic acid incorporation into non-saponifiable lipids and quantification of labeled cholesterol among its precursors. Synthesis was slow, in spite of a high 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA) activity and led to an accumulation of unexpected non-polar metabolites identified as C-30 sterones, but not C-27 sterol precursors. This supports a post-HMG-CoA regulation at the lanosterol demethylation step. Cholesterol biosynthesis was stimulated by cell culture in lipid depleted medium and was inhibited by pentadecane-2-one which acts mainly at two post-HMG-CoA steps: lanosterol demethylation and lathosterol isomerisation to cholesterol. A parallel pentadecane-2-one inhibition of cell growth was also observed, even when cells were cultured in the presence of whole serum. This indicates the existence of a relationship between endogenous cholesterol synthesis and cell growth and sheds additional light on the role of post-HMG-CoA regulation in this phenomenon.

Digitonin-precipitable sterols as a measure of cholesterol biosynthesis: contradictory results.

In experiments with 4 different types of cells, we evaluated the cholesterogenic activity by incorporation of 14C-acetic acid into cholesterol and digitonin-precipitable sterols. In every case, the cholesterogenesis appeared considerably faster when expressed as digitonid than when expressed as real cholesterol production, and sometimes the data obtained by the 2 methods were contradictory. Detailed analysis of both digitonid components and nonprecipitable radioactive metabolites showed that a very variable fraction of methyl sterols (including bifunctional methyl sterols) co-precipitates with the C-27 sterols. In cholesterol regulation studies and particularly when the cells exhibit a low cholesterogenesis, the digitonin method is unsuitable and can lead to erroneous interpretations.

DNA and cholesterol biosynthesis in synchronized embryonic rat fibroblasts. I. Temporal relationships between HMG-CoA reductase activity, sterol biosynthesis and thymidine incorporation into DNA.

Temporal relationships between hydroxymethylglutaryl-CoA reductase activity, biosynthesis of C27 sterols, and [3H]thymidine incorporation into DNA were studied in a rat embryo fibroblast cell line synchronized by double thymidine block and cultured in cholesterol-containing medium. Cyclic variations of HMG-CoA reductase activity and C27 sterols occurred, with two maxima in S and G2M phases; the relative shortness of the G1 phase (3 h) in these cells could be responsible for the shift of sterol synthesis in the S phase. No noticeable variation of the individual C27 sterols was observed during the entire cell cycle. In each experiment, there was a good linear correlation between HMG-CoA reductase activity and C27 sterol synthesis, but from one experiment to another, a given level of enzymatic activity led to varying levels of [2-14C]acetate incorporation into sterols. In our experimental conditions, total HMG-CoA reductase activity is measured, and the preceding observation could be explained by a varying degree of phosphorylation of the enzyme depending on the metabolic state of the cells at the start of the experiment. The cyclic variations of the enzyme activity seem to be due more to increased synthesis at given times of the cycle than to periodic dephosphorylation. We question the existence of a relationship between cell division and cyclic sterol synthesis occurring in cells cultured in cholesterol-containing medium.

Comparative study of the kinetics of 3-hydroxy-3-methylglutaryl coenzyme A reductase and [14C]-acetate incorporation into cholesterol in human lymphocytes stimulated by phytohemagglutinin or sterol efflux.

The time course of sterol biosynthesis was compared after two different stimulations in normal human lymphocytes: culture either in the presence of phytohemagglutinin (PHA) or in a lipid-depleted medium (i. e. a condition which produces sterol efflux). Stimulation by PHA gives rise to an acute and rapid response in 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and cholesterol synthesis. The sterol efflux induction produces a slower and milder response and the delayed sterol production consists mainly of lathosterol.

Squalene epoxidase, oxido-squalene cyclase and cholesterol biosynthesis in normal and tumoral mucosa of the human gastro-intestinal tract. Evidence of post-HHG CoA regulation.

Cholesterol biosynthesis was evaluated in different parts of the human gastro-intestinal tract by incorporation of labelled acetic or mevalonic acid into non-saponifiable metabolites and also by measuring squalene epoxidase activity, oxido=squalene cyclase activity and dihydrolanosterol demethylation rate. With respect to rat liver, whole cholesterol biosynthesis was generally low and we found an anatomic localization of activity towards the end of the gastro-intestinal tract. The results of analysis of mevalonic acid metabolites implied the existence of post-HMG CoA regulation. A comparison with the corresponding dat obtained from homologous cancerous tissues (inthe case of high cholesterogenic activity) did not show a greater activity in the tumor, in spite of higher levels of epoxidase concentration.

Squalene epoxidase and oxidosqualene lanosterol-cyclase activities in cholesterogenic and non-cholesterogenic tissues.

Squalene epoxidase and oxidosqualene lanosterol-cyclase activities have been studied in normal mammalian cholesterogenic and non-cholesterogenic tissues. This paper describes the kinetic conditions of measurement of these two enzymatic acitivities and their results. Oxidosqualene lanosterol-cyclase is a widespread enzyme, present in all cholesterogenic and non-cholesterogenic tissues. However, the level of squalene epoxidase is very low in non-cholesterogenic tissues. However, the level of squalene epoxidase is very low in non-cholesterogenic tissues. The effects of subcellular fractionizing and of the physico-chemical state of squalene incubated in vitro on squalene epoxidase activity are discussed.