Effect of an hdm-2 antagonist peptide inhibitor on cell cycle progression in p53-deficient H1299 human lung carcinoma cells.

The hdm-2 oncogene is overexpressed in several types of malignancies including osteosarcomas, soft tissue sarcomas and gliomas and hdm-2 has been associated with accelerated tumor formation in both hereditary and sporadic cancers. Among the other key binding partners, hdm-2 forms a complex with the tumor suppressor p53, resulting in a rapid proteasome-mediated degradation of the p53 protein. This positions the hdm-2-p53 complex as an attractive target for the development of anticancer therapy and recently the first small molecule hdm-2 antagonist has been reported. Development of hdm-2 antagonists is currently focused on malignancies containing a wild-type p53 genotype, which is the case in approximately half of human cancer indications. However, hdm-2 has also been implicated in oncogenesis in the absence of p53. We therefore studied the effect of hdm-2 antagonists in p53-deficient human H1299 lung carcinoma cells. The hdm-2 antagonistic peptide caused G1 cell cycle arrest, inhibited colony growth and induced expression of G1 checkpoint regulatory proteins, such as p21(waf1,cip1). These data demonstrate that hdm-2 regulates the G1 cell cycle checkpoint in a p53-independent manner, suggesting that hdm-2 antagonists represent a novel class of anticancer therapeutics with broad applicability towards tumors with different p53 genetic backgrounds.

Assay development for high throughput screening of p21(Waf1/Cip1) protein expression in intact cells using fluorometric microvolume assay technology.

The p21(Waf1/Cip1) protein represents a broad-acting cyclin-dependent kinase inhibitor that plays a key role in cell cycle regulation. Furthermore, p21(Waf1/Cip1) protein has been described as a direct participant in regulating genes involved in growth arrest, senescence and aging. In response to genotoxic insults (e.g., following chemotherapeutic treatment), p21(Waf1/Cip1) protein accumulates mainly through p53-mediated transcriptional activation and is also regulated at the post-transcriptional level. In tumor cells, p53 is frequently mutated leading to reduced p21(Waf1/Cip1) protein induction that may contribute to resistance to treatment by DNA-damaging agents. In order to identify compounds capable of restoring p21(Waf1/Cip1) protein level, we have developed a 96-multi-well plate-based high throughput screening assay in intact cells using the Applied Biosystems Fluorometric Microvolume Assay Technology (FMAT) macro-confocal system. Briefly, following incubation with test compounds, human MCF7 breast carcinoma cells were fixed and p21(Waf1/Cip1) protein was detected using anti-p21(Waf1/Cip1) monoclonal antibody and anti-mouse IgG conjugated to the red fluorescent dye Alexafluor 633. FMAT provides a set of raw images at a high magnification, in which fluorescence concentrated in a cell is detected as a specific signal. The mean fluorescence of a population of cells is calculated independently of the number of cells as with a classical FACS analysis. This is of particular interest for screening anticancer drugs that may affect cell number and therefore may impact on the readout. This assay was validated using reference compounds such as camptothecin and actinomycin D, known inducers of p21(Waf1/Cip1) protein.

SCAP ligands are potent new lipid-lowering drugs.

Upregulation of low-density lipoprotein receptor (LDLr) is a key mechanism to control elevated plasma LDL-cholesterol levels. Here we identify a new class of compounds that directly binds to the sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP). We show that a 14C-labeled, photo-activatable analog specifically labeled both SCAP and a truncated form of SCAP containing the sterol-sensing domain. When administered to hyperlipidemic hamsters, SCAP ligands reduced both LDL cholesterol and triglycerides levels by up to 80% with a three-fold increase in LDLr mRNA in the livers. Using human hepatoma cells, we show that these compounds act through the sterol-responsive element of the LDLr promoter and activate the SCAP/SREBP pathway, leading to increased LDLr expression and activity, even in presence of excess of sterols. These findings have led to the identification of a class of compounds that represent a promising new class of hypolipidemic drugs.

Multidrug resistance P-glycoprotein is not involved in cholesterol esterification.

The aim of the present paper is to reinvestigate the role of multidrug resistance P-glycoprotein MDR1 and MDR-associated protein (MRP1) in cholesterol esterification using well-characterized inhibitors. Using specific substrate efflux assay, we show that GF120918 (0.2 microM) and probenecid (5 mM) were specific inhibitors of MDR1 and MRP1, respectively. In HepG2 cells, neither of them affect the esterification of cholesterol derived from the uptake of cholesterol-rich lipoprotein, while both verapamil (100 microM) and progesterone (100 microM) were able to inhibit cholesterol esterification. Similar results were obtained with verapamil, progesterone, and GF120918 in the MDR1-overexpressing cells MCF7/ADR. The capacity of progesterone to reduce cholesterol esterification is not correlated with its ability to inhibit MDR1 but is rather due to direct inhibition of acyl-CoA:cholesterol acyltransferase (ACAT). We conclude that the esterification of cholesterol is not correlated with MDR1 or MRP1 activity, thus excluding their role in the intracellular transport of endocytosis-derived cholesterol.

Identification of domains in apolipoprotein B100 that confer a high requirement for the microsomal triglyceride transfer protein.

The microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apoB-containing lipoproteins. To investigate the role of MTP in lipoprotein assembly, we determined the ability of carboxyl-terminally truncated forms of apoB to be secreted from cells treated with the MTP inhibitor 4'-bromo-3'-methylmetaqualone (Benoist, F., Nicodeme, E., and Grand-Perret, T. (1996) Eur. J. Biochem. 240, 713-720). In Caco-2 and mhAT3F cells that produce apoB100 and apoB48, the inhibitor preferentially blocked apoB100 secretion. When the inhibitor was tested on McA-RH7777 cells stably transfected with cDNAs encoding human apoB100, apoB72, apoB53, apoB29, and apoB18, the secretion of apoB100, apoB72, and apoB53 was preferentially impaired relative to apoB48 and shorter forms. To delineate the region between apoB48 and apoB53 that has a high requirement for MTP, we used puromycin to generate a range of truncated forms of apoB in HepG2 cells. The secretion of apoB53 and longer forms of apoB was markedly affected by low concentrations of the MTP inhibitor (approximately 1 microM), whereas apoB51 and smaller forms of apoB were only affected at higher concentrations (> 10 microM). The size-related sensitivity to MTP inhibitor was not due to late processing or retention, since the same result was observed when nascent lipoproteins were isolated from the endoplasmic reticulum. The MTP inhibitor did not alter the density of the secreted lipoproteins, indicating that each apoB polypeptide requires a minimally defined amount of lipid to attain a secretable conformation. Our results suggest that the folding of the domain between apoB51 and apoB53 has a high requirement for lipid. This domain is predicted to form amphipathic alpha-helices and to bind lipid reversibly. It proceeds and is followed by rigid amphipathic beta-sheets that are predicted to associate with lipid irreversibly. We speculate that these domains enable apoB to switch from a stable lipid-poor conformation in apoB48 to another lipid-rich conformation in apoB100 during lipoprotein assembly.

Regulated turnover of a cell surface-associated pool of newly synthesized apolipoprotein E in HepG2 cells.

Newly synthesized apolipoprotein E (apoE) is incompletely secreted and partially degraded by hepatocytes. To evaluate current models concerning apoE's role in remnant lipoprotein clearance by the liver, we performed a detailed and quantitative pulse-chase analysis of apoE secretion, subcellular distribution, and proteolytic degradation by the human hepatoma cell line HepG2. Only about 35% of newly synthesized apoE were found to be secreted to the culture medium. As determined by a protease-protection assay, a substantial amount of newly synthesized apoE was transported to the cell surface, constituting more than half of the cellular apoE under steady-state conditions. A subpopulation representing almost 40% of newly secreted apoE was rapidly rebound to the cell surface, indicating a dynamic equilibrium between cell surface and secreted apoE. These pools of newly synthesized apoE were subject to proteolytic turnover that occurred in lysosomes, presumably after re-endocytosis. We found that the proteolytic turnover of cell surface and secreted apoE was regulated by the availability of apoE ligands, being almost completely suppressed by the presence of lipoprotein-containing human serum or isolated lipoproteins, namely LDL and HDL. The characteristics of regulated degradation of cell surface apoE shed new light on potential physiological functions of this pool of apoE. Our results provide evidence that hepatocytes are capable of forming a large pool of cell surface-associated apoE, thereby supporting the previously proposed secretion-recapture model for apoE.

Co-translational degradation of apolipoprotein B100 by the proteasome is prevented by microsomal triglyceride transfer protein. Synchronized translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride transfer protein.

We studied the effect of inhibition of microsomal triglyceride transfer protein (MTP) on apolipoprotein (apo) B100 translation and secretion using HepG2 cells. The MTP-mediated lipid transfer activity was reduced using a specific MTP inhibitor. ApoB100 translation was synchronized by treatment with puromycin prior to L-[35S]methionine pulse-chase labeling. During the first 4 min of chase, synthesis of apoB polypeptides the size of 100-200 kDa was insensitive to the inhibitor, suggesting that inhibition of MTP did not affect the initiation of apoB100 translation. After 15 min of chase, the 100-200-kDa species were chased into polypeptides larger than 320 kDa (i.e. apoB65 or 65% of full-length apoB100) in both control and inhibitor-treated cells. However, the amount of these polypeptides decreased (by 36% for apoB65-75, by 64% for apoB75-85, by 76% for apoB85-95, and by 77% for apoB100) upon MTP inhibition. No accumulation of smaller polypeptides was observed, but total immunoprecipitable apoB radioactivity was decreased suggesting that apoB could undergo co-translational degradation when MTP activity was reduced. Inhibitors of the multicatalytic proteinase complex (proteasome) such as lactacystin or MG-115 could prevent apoB co-translational degradation. Nevertheless, MG-115 could not avoid the MTP inhibitor decreasing apoB100 secretion but rather induced the accumulation of secretion-incompetent apoB100 in the cell. These results indicate that MTP activity is required during the elongation of apoB100 polypeptides, particularly at the sequences downstream of carboxyl terminus of apoB65. Co-translational degradation might constitute a more general mechanism of early quality control for large or complex proteins.

ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools.

Triglycerides (TGs), cholesteryl esters (CEs), cholesterol, and phosphatidylcholine have been independently proposed as playing regulatory roles in apoB-100 secretion; the results depended on the cellular model used. In this study, we reinvestigate the role of lipids in apoB-100 production in HepG2 cells and in particular, we clarify the respective roles of intracellular mass and the biosynthesis of lipids in the regulation of apoB-100 production. In a first set of experiments, the pool size of cholesterol, CEs, and TGs was modulated by a 3-day treatment with either lipid precursors or inhibitors of enzymes involved in lipid synthesis. We used simvastatin (a hydroxymethylglutaryl coenzyme A reductase inhibitor), 58-035 (an acyl coenzyme A cholesterol acyltransferase inhibitor), 5-tetradecyloxy-2-furancarboxylic acid (TOFA, an inhibitor of fatty acid synthesis), and oleic acid. The secretion rate of apoB-100 was not affected by the large modulation of lipid mass induced by these various pre-treatments. In a second set of experiments, the same lipid modulators were added during a 4-hour labeling period. Simvastatin and 58-035 inhibited cholesterol and CE synthesis without affecting apoB-100 secretion. By contrast, treatment of HepG2 cells with TOFA resulted in the inhibition of TG synthesis and apoB-100 secretion. This effect was highly specific for apoB-100 and was reversed by adding oleic acid, which stimulated both TG synthesis and apoB-100 secretion. Moreover, a combination of oleic acid and 58-035 inhibited CE biosynthesis and increased both TG synthesis and apoB-100 secretion. These results show that in HepG2 cells TG biosynthesis regulates apoB-100 secretion, whereas the rate of cholesterol or CE biosynthesis has no effect.

Microsomal triacylglycerol transfer protein prevents presecretory degradation of apolipoprotein B-100. A dithiothreitol-sensitive protease is involved.

The role of microsomal triacylglycerol transfer protein (MTP) in the secretion of apolipoprotein B-100 (apoB-100) has been studied using an inhibitor of MTP: 4'-bromo-3'-methylmetaqualone. In vitro, this compound inhibits trioleoylglycerol transfer between lipid vesicles mediated by MTP with an IC50 of 0.9 microM whereas it does not inhibit the lipid transfer mediated by the cholesteryl ester transfer protein. In HepG2 cells, 4'-bromo-3'-methylmetaqualone inhibits the secretion of apoB-100 with an IC50 of 0.3 microM, without affecting the secretion of several other proteins like apoA-I or albumin. Moreover, there is no accumulation of apoB-100 in treated cells. Oleic acid, which increases apoB-100 secretion, only slightly modifies the IC50 of 4'-bromo-3'-methylmetaqualone (0.5 microM). The latter has no effect on the synthesis of major lipids within the cell, but decreases the secretion of triacylglycerol into apoB-100-containing lipoproteins. Pulse/chase experiments reveal that 4'-bromo-3'-methylmetaqualone acts on apoB-100 production either at the co-translational or post-translational level. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal does not protect apoB-100 from the 4'-bromo-3'-methylmetaqualone effect but seems to be involved in a later step of apoB-100 intracellular degradation. By contrast, dithiothreitol can totally reverse the effect of the MTP inhibitor on apoB-100 production. The mechanism of MTP-mediated lipid assembly with apoB-100 is discussed.

A sensitive method to analyze in vitro secretion of lipoproteins: distribution of apolipoproteins is modulated by oleic acid in HepG2 cells.

Lipoprotein metabolism can be studied by the analysis of lipoprotein production in cell culture. An inherent problem in such an analysis is the low concentration of lipoproteins in culture supernatants. The difficulty comes from the fact that the samples must be concentrated prior to any analysis. The concentrating methods (e.g., dialysis or ultrafiltration) induce a heterogeneous loss of components. In order to minimize these losses, we have developed a sensitive three-step method to analyze the distribution and the amount of apolipoproteins in the different classes of lipoproteins secreted by the human hepatoma cell line HepG2. Cells were labeled with [14C]acetate and [35S]methionine for 4 h in the presence of 0.08 mM BSA, complexed or not, with 0.75 mM oleic acid. The 14C-radiolabeled cellular lipids were extracted and analyzed by thin-layer chromatography and the secreted lipoproteins were analyzed by the following three-step method. First, the lipoproteins were isolated by flotation ultracentrifugation. Second, total lipoproteins were directly applied to native agarose-acrylamide gel electrophoresis in order to separate lipoproteins with respect to their diameter. After migration, the gel was sliced and each fragment was eluted in a buffer containing sodium dodecyl sulfate and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This allowed evaluation of the proportion of apolipoproteins in lipoproteins. Oleic acid (0.75 mM) increased the rate of triglyceride biosynthesis and apoB-100 secretion by 1.7-fold and 2.4-fold, respectively. Moreover, oleic acid treatment modified the profile of secreted lipoproteins. Oleic acid-treated cells secreted more apoB-100 within VLDL than control cells.

In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative.

N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]- phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) has been selected from a chemical program aimed at identifying an optimized inhibitor of multidrug resistance (MDR). The potency of GF120918 is assessed by dose-dependent sensitization of CHRC5, OV1/DXR and MCF7/ADR cells to the cytotoxicity of doxorubicin and vincristine respectively: GF120918 fully reverses multidrug resistance at 0.05 to 0.1 microM and is half maximally active at 0.02 microM. The spectrum of drugs sensitized by GF120918 coincides with those having the classical MDR phenotype. In CHRC5 cells, 0.01-0.1 microM GF120918 enhances the uptake of [3H]daunorubicin and blocks the efflux from preloaded cells. It is also shown that GF120918 is still active several hours after being taken away from the culture medium showing that it is not, like verapamil, effluxed rapidly by P-glycoprotein. GF120918 effectively competes with [3H]azidopine for binding P-glycoprotein, pointing to this transport membrane protein as its likely site of action. After i.v. administration to mice, GF120918 penetrates thoroughly various organs that have a tissue level/blood level ratio above 10. It is eliminated from organs and blood with a half-time of approximately 2.7 h. It is well absorbed after p.o. administration. In mice implanted i.p. with the MDR P388/Dox tumor, a single i.v. or p.o. dose of GF120918 restores sensitivity of the tumor to a single i.p. dose (5 mg/kg) of doxorubicin administered 1 h later. A statistically significant effect is observed at 1 mg/kg GF120918 i.v. and maximal effect is reached at 5 mg/kg. Similarly, whereas neither drug alone is effective, GF120918 (10 mg/kg i.p.) associated with doxorubicin (5 mg/kg i.p.) inhibits the growth of the moderately MDR C26 tumor implanted s.c. as assessed by tumor size at day 19. GF120918 does not modify significantly the distribution or the elimination of doxorubicin in mice ruling out the possibility that the antitumor effects seen might be explained by pharmacokinetic interactions.

The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C.

Staurosporine is the most potent inhibitor of protein kinase C (PKC) described in the literature with a half-maximal inhibitory concentration (IC50) of 10 nM. Nevertheless, this natural product is poorly selective when assayed against other protein kinases. In order to obtain specific PKC inhibitors, a series of bisindolylmaleimides has been synthesized. Structure-activity relationship studies allowed the determination of the substructure responsible for conferring high potency and lack of selectivity in the staurosporine molecule. Several aminoalkyl bisindolylmaleimides were found to be potent and selective PKC inhibitors (IC50 values from 5 to 70 nM). Among these compounds GF 109203X has been chosen for further studies aiming at the characterization of this chemical family. GF 109203X was a competitive inhibitor with respect to ATP (Ki = 14 +/- 3 NM) and displayed high selectivity for PKC as compared to five different protein kinases. We further determined the potency and specificity of GF 109203X in two cellular models: human platelets and Swiss 3T3 fibroblasts. GF 109203X efficiently prevented PKC-mediated phosphorylations of an Mr = 47,000 protein in platelets and of an Mr = 80,000 protein in Swiss 3T3 cells. In contrast, in the same models, the PKC inhibitor failed to prevent PKC-independent phosphorylations. GF 109203X inhibited collagen- and alpha-thrombin-induced platelet aggregation as well as collagen-triggered ATP secretion. However, ADP-dependent reversible aggregation was not modified. In Swiss 3T3 fibroblasts, GF 109203X reversed the inhibition of epidermal growth factor binding induced by phorbol 12,13-dibutyrate and prevented [3H] thymidine incorporation into DNA, only when this was elicited by growth promoting agents which activate PKC. Our results illustrate the potential of GF 109203X as a tool for studying the involvement of PKC in signal transduction pathways.

Antimetastatic effect of immunomodulators from Nocardia opaca in mice and rats activation of peritoneal macrophages by these fractions.

Nocardia delipidated cell mitogen (NDCM), a particulate fraction prepared from Nocardia opaca, injected i.p. in an oil/water emulsion to F6 rhabdomyosarcoma-bearing rats, inhibited the development of pulmonary metastases; 6 out of 10 rats were protected. Repeated i.p. administration of emulsified NDCM and of two other compounds, a Nocardia water soluble mitogen (NWSM a hydrosoluble fraction) and purified cell walls (CW, an insoluble macromolecular fraction) in Lewis lung carcinoma (LLC)-bearing mice resulted in a significant reduction of lung metastases. The efficiency of these fractions was enhanced by association with monokines. A combination regimen of NDCM, NWSM, and CW (100 micrograms/0.1 ml) and monokines (0.1 ml), injected i.p. in LLC-bearing mice, yielded a greater antimetastatic effect than either therapy alone. Peritoneal macrophages from mice which had been injected i.p. with NWSM or CW, when triggered either by TPA (tetradecanoyl phorbol acetate) or by zymosan, released large quantities of hydrogen peroxide and had a high rate of glucose consumption. These macrophages were activated as judged by their cytostatic activity against syngeneic P815 mastocytoma growth; they expressed biochemical markers which have been reported to characterize the activated state. Incubation of thioglycollate-elicited peritoneal macrophages with NWSM, and monokines for 72 h resulted in a cytotoxic activity against labeled LLC cells; addition of macrophage activating factor significantly increased the cytotoxic capacity of these macrophages. In view of this we postulate that the antimetastatic effect of soluble and insoluble N. opaca fractions and monokines might be mediated by activated peritoneal macrophages.

Modifications induced by activation to tumor cytotoxicity in the protein secretory activity of macrophages.

After biosynthetic radiolabeling, polypeptides secreted by macrophages were analyzed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate and fluorography. Comparative studies of various macrophage populations (inflammatory macrophages, primed and activated macrophages, resident macrophages) showed that secretory activity is elaborately controlled according to macrophage developmental and functional state. When young macrophages activated to cytotoxicity against tumor cells by an appropriate eliciting agent and culture in the presence of lipopolysaccharide were compared to mature resident macrophages devoid of antitumor activity, the following changes were observed in the protein secretion phenotype: (1) New polypeptides are expressed: the intensity of four bands was increased more than five-fold on one-dimensional gels and 17 new spots were detected on two-dimensional gels. (2) The production of several major polypeptides is repressed: six bands were strongly decreased on one-dimensional gels, and 11 major spots (or groups of spots) were lost on two-dimensional gels. (3) The charge microheterogeneity pattern exhibited by several polypeptides on two-dimensional gels is shifted towards less acidic species.

Macrophage activation by trehalose dimycolate requirement for an expression signal in vitro for antitumoral activity; biochemical markers distinguishing primed and fully activated macrophages.

It was possible to define the effects of trehalose dimycolate (TDM), a glycolipid extracted from Mycobacterium tuberculosis, on mouse peritoneal macrophages more precisely using endotoxin-free culture conditions. TDM-elicited macrophages, when assayed in vitro in the absence of endotoxin, were unable to limit tumor growth; however, after a short treatment (4 h) with low doses of lipopolysaccharide (LPS; 1-10 ng/ml), they exhibited a strong cytostatic capacity against P815 mastocytoma cells. Thus, TDM injected in vivo did not activate macrophages fully but it primed them to respond in vitro to low doses of LPS, which provided the final stimulus for activation to antitumor competence. Macrophages elicited by an injection of killed group C Streptococci were also in a primed state; in contrast, thioglycollate-elicited macrophages were in a nonreceptive state. Besides LPS, concanavalin A (5 micrograms/ml), MDP (0.2-1 microgram/ml) and the ionophore A23187 (5 microM) can deliver the activation signal to TDM-primed macrophages. Primed macrophages were found to express several biochemical markers previously described as specific for activated macrophages (low levels of alkaline phosphodiesterase and beta-galactosidase, for example) and, although they were not cytotoxic for tumor cells, they had the capacity to release large amounts of H2O2. However, when pulsed by LPS or MDP, primed macrophages responded by further modifications in their metabolism: the rate of glucose consumption and the labeling of glycoproteins by D-[2-3H]mannose were greatly increased and the secretion of a polypeptide of 22 kDa was enhanced. The activation-associated biochemical markers are thus acquired in two steps. The ability to produce activated oxygen species is expressed earlier than the antitumoral activity.