Involvement of tyrosine kinase activity in the low-density lipoprotein receptor expression in human lung adenocarcinoma cell line A549

Bkwhimie ( 1996) 78, X74-88 1

0 Soci&? franqaise de biochimie et biologie molt?culaire / Eisevier. Paris

N Gueddari, G Favre, C Marmouget, G Soula, F Le Gaillard* Lahosatoire de Cihlap CIIThcraptvltiqw, EAIUPRES, UniversitP Paul Sahatier (Tohu~st). FacultP des Scierwes Pl~ar.nla~elrticitles et Centre Claudius Regaud, 20-24. rue du Pant-St-Pierre, 3 IO52 Touhrse cede.\-.France

(Received I2 March 1996: accepted 28 June 1996) Summary - In common with other tumour cell lines but in contrast to normal cells, the human adenocarcinoma cell line AS49 showed a ?nd metabolism (sum of internalised and degraded LDL) biphasic regulation of the LDL receptor activity during growth: both LDL bindin, 0 L increased during the log exponential growth phase and decreased when the cells approached confluence. This period of increasing LDL receptor activity coincided with a high resistance to cholesterol down-regulation which suggested a sterol-independent pathway of stimulation. Since A549 cells have an autocrine loop of growth factors, two of which have tyrosine kinase activity, the LDL receptor activity was tested in the presence of the tyrosine kinase inhibitor, genistein. When cells were incubated in the absence of cholesterol (LPDS medium), the inhibition that occurred was two-fold higher during the exponential growth phase than during the confluent phase. Moreover, the residual LDL binding and metabolism after genistein inhibition were completely resistant to down-regulation by cholesterol only during the growth phase. When cholesterol was present (FCS medium), inhibition was observed only during the growth phase. The inhibition of LDL receptor activity by genistein was found to be the result of a loss in the number of LDL binding sites, while the dissociation constant was not affected. This loss was accompanied by a disappearance of mRNA as shown by RNase mapping. By comparison, LDL receptor activity of normal cells (fibroblasts) was also affected by genistein during the exponential growth phase but was much more cholesterol-dependent. Taken together, these results suggest that the tyrosine kinase pathway is essential to up-regulate LDL receptor expression in highly dividing cells and particularly in tumour cells in which the sterol regulation is deficient. LDL binding sites i LDL receptor gene expression I LDL receptor modulation I lung adenocarcinoma tyrosine kinuse

Introduction There are two ways by which mammalian cells obtain the cholesterol necessary for the synthesis of membranes. This can be by &J now synthesis, the rate-limiting step of which is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, or by the uptake of lipoproteinderived cholesterol r?iu the low density lipoprotein receptor (LDL receptor). Cells usually maintain a low rate of cholesterol synthesis and rely predominantly on the LDL receptor pathway for their cholesterol needs. It has also been shown that synthesis of the LDL receptor is subject to feed-back inhibition by intracellular cholesterol [I]. Indeed the incubation of different cell types in the presence of LDL cholesterol results in a decrease of the LDL receptor gene transcription [2].

*Correspondence and reprints Abbreviations: FCS, foetal calf serum; HMG-CoA, 3-hydroxy-3methylglutaryl coenzyme A; LDL, low-density lipoproteins; LDLR, low density lipoprotein receptor; LPDS, lipoprotein deficient serum.

cell line I A5

It has been reported that some malignant cell lines have higher LDL receptor activity than the corresponding normal cells 13.4). LDL uptake has been shown to be higher in lung tumour tissue than in the corresponding normal tissue [5]. Similarly, we have shown that in the presence of LDL-cholesterol, the human adenocarcinoma cell line A549 expressed a high level of LDL receptors compared with normal fibroblasts [6]. The reasons for this increase in LDL receptor activity and/or expression in tumour cells and tissues are not clear: they could be metabolic adaptation to excessive cholesterol requirement and/or modification directly related to cellular transformation. This abnormality could also be due to an alteration in the regulation of the sterol-dependent expression of LDL receptor and/or the involvement of a sterol-independent mechanism. The LDL receptor activity is regulated by cell density. It has been shown to be inversely correlated to cell density in fibroblasts [7, 81, smooth muscle cells [9] and endothelial cells [lo]. In fibroblasts, the decline in LDL receptor activity when cell density increases has been attributed to the decreased percentage of dividing cells [8, I I]. In several carcinoma cell lines, LDL receptor activity increases during growth phase and decreases in quiescent cells [ 121.

boratories-France. SPerilising filter units (Miller; 0 from illipore. Genistein was obtained from IOd All other reagents were from Sigma or Merck (France).

ary to changes in cellular cholesterol content or whether they act as primary effecters. wever, conditions have been described for some of the agents in which the increase in LDL receptor activity appears to be indep of cell cholesterol needs. The level of mRNA for L epG2 cells by a cytokine (oncostatin ate of the sterol repression [IS]. This is dependent on two cellular events: ein tyrosine phosphorylation; and ii) induction of a nuclear signal transducer (Egr-1) resulting from early biochemical events generated at the cell membrane [ 191. We have previously shown that, compared with fibroblasts, the LDL receptor expression in the human adenocarcinema cell line A549 is somewhat resistant to down-regulation by EDL-cholesterol [6]. This cell line produces several growth factor!: such as insulin-like growth factor (IGF 1 ), transforming growth factors a and p (TGF 01 and p), of which two are known to stimulate tyrosine kinase activity (TGF 01and IGF 1) [20]. The effects of cell density on LDL metabolism by tumoural cell lines might be mediated by cellular autocrine factors that are able to modul LDL receptor expression but very little is known about mechanism. The experiments reported here were designed to investigate the effects of A549 cell density on the regulation of their LDL receptor activity. In addition, to obtain further insights into the regulation of LDL receptor gene expression in these cells by tyrosine kinase activity, we investigated the effect of genistein, a tyrosine kinase inhibitor, on LDL receptor expression and activity when cells are in growth and in confluence phases. Materials and methods Materials

Cultured fibroblasts were established from explants of human skin as previously described [21 J. A549 cells (ATCC CCL 185) were obtained from the American Type Culture Collection (Rockville, MD). The cell line, which possesses epithelial-like morphology, was initiated through explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male. Sodium 1?5I iodide (100 mCi/mL) was obtained from Amersham (France). Foetal calf serum (FCS), powdered Dulbecco’s modified Eagle’s medium (DMEM), RPM1 1640 and Dulbecco’s phosphate buffered saline (PBS) were obtained from Gibco (France). Thrombin 500 was from Houd&La-

uman LDL (6 = 1.019-l .05O g/mL), DL (6= 1.063-1.210 g/mLj, and human LPDS serum (6 > 1.25 g/mL) were isolated from fresh human plasma of healthy donors by ultracentrifuge flotation, in KBr density gradients using a vertical ultracentrifugation rotor (VTi-50, Beckman, France) 1221. After extensive dialysis against 0.3 mM EDTA. 0.15 M N&I, (pH 7.4j, lipoproteins and LPDS were sterilised by filtration through membrane filter (Millipore 0.45 pm pore size filter) and stored at 4°C (up lo 3 weeks} for the lipoproteins and -20°C for up to 3 months for LPDS. LDL were iodinated by the iodine monochloride method of Mac Farlane as modified by Bilheimer et al [23]. The final specific activities varied between 149 and 243 cpm per ng of protein. Protein concentrations were determined by the method of Lowry with bovine serum albumin as standard: hereafter the concentrations of lipoproteins refer to their protein content.

Cells were grown in plastic tissue culture flasks (25 cm’) in RPMI 1640 and in DMEM for A549 cells and human skin fibrobllasts respectively. The media contained 0.002 M glucose, 0.24 M CO3 and were supplemented with 10% (v/v) heat inactivated FCS (56OC, 30 min). The cell cultures were kept at 37°C in humiditied incubators equilibrated with 5% CO?, 95% air. The doubling times of the A549 cells and fibroblasts, in the exponential growth phase, were 24 h and 48 h respectively. The A549 cells were subcultured twice a week and the fibroblasts every week.

Before each experiment the cells were trypsinised in the exponcnlial growth phase from stock flasks and seeded (day 0) in 6-well multidishes (35 mm diameter). The binding, internalisation and degradation of ‘zsI-LDL at 37°C and the binding at 4°C were measured as described by Goldstein and Brown [24]. For measurement of binding and metabolism (sum of internalisation + degradation) of l%labelled LDL, the medium of each dish was removed and the cell monolayers were washed twice with warm PBS (37°C). Then I mL of LPDS medium containing the indicated concentrations of IWLDL (15 l.@mL), in the presence or absence of a 40-fold excess of unlabelled LDL, was added to each dish to initiate the experiment. After 5 h of incubation at 37OC, the cells were placed on ice. The medium of each dish was removed, an aliquot taken for the measurement of trichloroacetic acid-soluble degradation products of IZsILDL according to the method described by Goldstein and Brown [ 241. After the cells were washed, each dish received 1 mL of ice-cold medium containing 50 mM NaCl and 10 mM HEPES (pH 7.4) to which heparin (final concentration: 10 mg/mL) was added. The dishes were placed in a 4°C cold room for 60 min after which the medium was removed and a 0.5 mL aliquot counted for its content of 1’5-1radioactivity (heparin-releasable lz”I-LDL) [25]. The cells were then dissolved by incubation at room temperature for at least 60 min in 1 mL of 0.5 M NaOH. One aliquot (0.5 mL) was counted

876 to determine the intemalised W-LDL (heparin-resistant W-LDL) and another ahquot was used to determine the cell protein content. For the measurement of binding of izsl-labelled LDL at 4OC, the cells were placed on ice for 30 min, washed with PBS and placed on ice for 60 min in RPM1 1640 containing 1% BSA, 20 mM HEPES (pH 7.4). The medium was removed, and the cells were incubated in the same medium containing increasing concentrations of i”sI-LDL for 2 h in the presence or in absence of a 40-fold excess of unlabelled LDL. After the cells had been washed, the binding and cell protein content were determined as described above. Specific LDL receptor mRNAanalysis Extraction of RNA A549 cells were plated at 8 x 10s cells per 80 cm* flask. The total RNA was extracted from the cells according to the method described by Chomczinsky and Sacchi [26]. Briefly, the cells were washed in cold PBS without any RNase, harvested in the same buffer, centrifuged (8000 rpm, 5 min) and resuspended in 4 M guanidinium isothiocyanate, 25 mM citric acid (pH 7) solution containing 0.1 M emercaptoethanol and 0.5% sarcosyl. RNA was extracted from protein and DNA by adding isoamylic alcohol, sodium acetate pH 4 (final concentrations of 0.2% and 0.2 M respectively) and a volume of water-saturated phenol. The aqueous phase was then precipitated twice by one volume of isopropanol at -20°C for 2 h. After centrifugation at 13 000 g for 10 min, the pellet was resuspended in 0.5% SDS, heated for 5 min at 65°C and stored at -8OOC until use. The amount of RNA obtained was measured by the absorbance at 260 nm and the integrity of the material was checked by electrophoresis on 0.8% agarose gel. Rnase mapping In our hands and according to others [27], the RNase mapping method produces less non-specific hybridisation than Northern blot. The probes used for the RNase mapping experiments were the products of in vitro transcription in the presence of .lzP UTP (3000 Ci/mmol, ICN) of LDLR-cDNA and 36 B4 cDNA which had been cloned in pKS plasmid containing the double promotor T3 and T7 in opposite directions. The fragment used for LDL probe was the 380 bp fragment BarnHI-EcoRI of the complete LDLR cDNA and for the 36 84 probe, the fragment BarnHI-/find111 (650 bp) kindly provided by P Chambon. This latter probe was used as a constant probe as reported by Masiakowskiet al (281. Total RNA was incubated in the presence of LDLR and 36 84 probes (5 x 105 cpm) in hybridisation buffer (10 mM Tris, pH 8.6,5 mM EDTA, 0.3 M NaCI) for 16 h at 55°C. Then, non-hybridised material was digested for 2 h at 37°C in the presence of RNase A (4 pglmL) (Sigma) and RNase Tl(7 ug/mL) (Boerhinger) in a 10 mM Tris (pH 8.6) buffer with 0.3 M NaCl and 50 mM EDTA followed by incubation in presence of lOOug/mLof proteinase K (Boerhinger)

for 15min at 37°C.The hybrids were then separatedfrom the proteins by adding one volume of a phenol-chloroform solution (l/l); the aqueous phase was precipitated in the presence of 50 pg/mL of tRNA as a carrier and 0.5 M LiCl in one volume of isopropanol.The hybrids were resuspended in the electrophoresis buffer (80% deionised formamide, 0.2% bromophenol blue, 0.2% xylene cyanol) and heated to 95°C for 2 min before analysis by electrophoresis on a denaturing polyacrylamide gel (8 M urea, 5% acrylamide). After fixation in a 10%acetic acid, 10% methanol solution, the gel was dried and autoradiographed using Hyperfdm MP (Amersham).The intensity of the

band corresponding to the messenger was determined with a scanning densitometer (Sebia).

esults irz e.qwnential growin,~ A549 Cells, the LDL rec’eptoractivity increases and is resistant to cholestesol down-wgum’atio~ The LDL receptor activity was expressed as LDL binding capacity and LDL metabolism (sum of intemalisation and degradation) with respect to cell density. The rates of LDL binding and metabolism in A549 cells increased with cell density to reach maximum values when cells were seeded at 5 x 104 per well. Thereafter, the rates of binding and metabolism decreased as cell density increased: bound and metabolised LDL values were respectively 2.5- and 2.2-fold lower when the cell density was ten fold higher (50 x 104 seeded cells per well) (fig 1). The observed increase in L L receptor activity was related to cells in log-exponential phase of growth. In contrast, when cells were seeded at a ten-fold higher quantity, they approached confluence when the experiments were performed. Determination of LDL binding and metabolism in the presence or absence of cholesterol (FCS or LPDS medium) during these two growth phases were determined. The resistance to down-regulation by cholesterol was shown to be higher during the growth phase than during confluence phase (control values of table I).

OLI

10

I

100

1ooo

Seeded cells per well x lo” Fig 1. Effect of cell density on binding and metabolism of 12’I-LDL in A549 cells. The cel!j were seeded in 35-mm diameter dishes at different densities in 10% FCS containing medium. At day 3, they were incubated fys,5 h at 37°C in medium containing 5% LPDS and 15 pg/mL of I-LDL (105 cpm/ng) with or without a 40-fold excess of uniabelled LDL. The specific binding (0), andlgftabolism (sum of intemalisation plus degradation) ( -LDL were determined as described in Materials arzd methods. The results represent means f SD of two independent experiments. Assays were carried out in triplicate for each experiment.

877

were carried

out in the presence ). A significant inned in growing cells but

Control + genistein Residual activity (%)

57.0 * 2.1 21.9+ 1.1

35 -t 4.2 27.6k2.2

705 + 85 34Ort72

580 k 42 429k3l

(38%)

(79%)

(48%)

(74%)

483 +22 373 -t 15

298 k 22 302f 12

Corrflrrerrt Control + genistein Residual activity (%)

72.3 f5.3 46.1 k3.4 (64%)

l6.63- 1.4 17.4+ 1.6 (100%)

(77%)

medium since residual s were respectively 79% and 74% for binding and metabolism (table I). hatever the inhibition conditions, the obtained percentage values of the residual activity were similar for both binding and metabolism. It seems therefore that the decrease in activity is due to a decrease in LDL binding. Tht analysis of binding parameters (number of sites and affinity) and assay of mRNA level were then performed. The decreased LDL receptor actil+ty, owing to genisteirl, is related to a decrease in m-responding mRNA lel*el

(100%)

The cells were seeded in dishes (35 mm) at density 10” or 5 x 10’ in FCS-containing medium. After 2 days of growth, the cells were washed with PBS and incubated in 10% FCS medium or 5% LPDS medium for 8 h. Genistein (40 pg/mL) or DMSO (6 pL/mL) were then added to cells and incubations were extended for 16 Il. The binding and metabolism of ““I-LDL were determined as described in Materials and methods. The results represent the means zkSD of two independent experiments. Assays were carried out in triplicate in each experiment.

Binding parameters of LDL receptor were determined by Scatchard analysis. It was carried out with t?sI-LDL at 4°C after a 16 h preincubation of the cells with genistein (40 mg/mL) in LPDS containing medium (fig 3). The dissociation constant of cells incubated with genistein (Ko = 14.3 k- 2.3 nM) and that of control cells

m

The increase in both LDL receptor activity and resistance to down-regulation by cholesterol in highly dividing cells suggested sterol-independent mechanism. Since A549 cells have an autocrine loop of growth factors, two of which have Tyr kinase activity, we tested LDL receptor activity in the presence of genistein. Genistein decreases

LDL receptor- actirity in AS49 cells

First of all, LDL receptor activity was determined with varying concentrations of genistein. The levels of LDL binding and metabolism by A549 cells, in LPDS conditions, decreased with an increase in genistein concentration (fig 2). Since a plateau for bound W-LDL was reached with a concentration of 40 mg/mL of genistein, this concentration was used for further experiments. The effect of genistein was determined in LPDS conditions for both exponentially growing cells and confluent cells. The residual LDL receptor activity, expressed as a percentage of the control value without genistein, was found to be lower in exponentially growing cells for both LDL binding and metabolism (table I). The percentage ratio (growing cells/confluent cells) is 0.6 for both LDL binding and LDL metabolism. The high inhibition, herein observed

0

_I, 0

I

IO

I

I

I

I

20

30

40

50

JO 60

Genistein (ug/ml) Fig 2. Effect of increasing concentrations of genistein on binding and metabolism of ““I-LDL in exponential growing A549 cells. The A549 cells were seeded (day 0) at lo5 cells per well (35 mm diameter). At day 2, the cells were washed with PBS buffer, preincuhated at 37°C in 5% LPDS medium for 8 h and then incubated in the same medium supplemented with increasing concentrations of gnistein for 16 h. The specific binding (0) and metabolism ( ‘I-LDL were determined as described in Materials and methods. The results represent means f SD of two independent experiments. Assays were carried out in triplicate for each experiment.

878 activation of the gene lranscription degradation.

or inhi

In order to investigate if the observed regulation of LDL receptors by Tyr kinase is a characteristic of tumour cells, we determined the LDL receptor activity when normal cells (fibroblasts) were exposed to genistein during their exponential growth phase. Incubation of fibroblasts with genistein was carried out in the same conditions as for A549 cells. An inhibition of LDL receptor activity occurred; it was higher in LPDS medium than in FCS medium (table II). This result was similar to the one obtained with A549 cells. Therefore, the inhibition by genistein seems to be more a characteristic of proliferating cells than of tumour cells.

0

10

30

20

%LDL

40

iseussion

(pghl)

Fig 3. Effect of genistein on ‘“‘I-LDL binding at 4°C in A549 cells. The A549 cells were seeded at day 0 at lo” cells per dish (35 mm diameter). At day 2, the cells were washed with PBS and preincubated at 37°C in 5% LPDS medium for 8 h and then incubated in the same medium supplemented with 40 j@rnL of genistein ( or 6 pL/mL of DMSO (0) for 16 h. After the cells had been washed with PBS buffer, they were incubated for 30 min in ice-cold medium containing SO mM NaCI and 20 mM HEPES (pH 7.4) then for 2 h at 4OC in t,Q? same medium in the presence of increasing concentrations of . I-LDL ( I SOcpm/ng) with or without 40-fold cxcw of unlabtj&d LDL. The cells were wasfjcjf and the amount of surface bound _*I-LDL (hcparin releasable “- I-LDL) was dctcrmined as described in iWcctc~kr1.s trrld twhods. Inset: Scatchard graph. The results represent means f SD of two independent experiments. Assays were carried out in triplicate for each cxpcriment.

The inverse relationship between LDL receptor activity and cell density has been previously described in normal fibroblasts [ 111. The increase of LDL receptor activity that we observed in growing A549 cells, appeared to be a characteristic of tumour cell lines. Indeed, similar results have been shown in some gynaecological cancer cell lines [ 121. The I-IMG CoA reductase activity, a key enzyme in cholesterol biosynthesis is higher in A549 cells than in normal cells [29] and it has shown a similar curve profile in relation to cellular density in A549 cells (unpublished results). Concurrently, an increase in acyl cholesterol acyltransferase (ACAT) activity, the choiesterol esterification enzyme, was found in confluent phase (data not shown). It is therefore

Table II. Effects of genistein on LDL binding and metabolism in normal fibroblasts in exponential growing conditions. 11-.__1_(Ku = 17.0 f 2.6 nM) were quite similar. In contrast, the number of sites in treated cells decreased from 147 f 10.6 ng/mg cell protein to 67.5 f 9.2 ng/mg cell protein. Thus, the observed decrease in 12%LDL binding could be entirely accounted for by a decrease in the number of LDL receptor sites number on the cell surface. The question then arises to know whether this observed low number of LDL receptors is the result of a decrease in mRNA level. This was therefore assayed after genistein treatment. When AS49 cells were deprived of exogenous cholesterol by substituting FCS for LPDS in culture medium for 24 h, there was a two-fold increase in the level of

LDL receptor mRNA. If genistein was added during this 24-h incubation as previously described, a disappearance of mRNA was observed (fig 4). Therefore, the regulation of LDL receptor by Tyr kinase activity seems to be related to

LDL binding

LDL nwtuhoiisnr

nglnig wll ptwteiti (70) LPDS

-___

FCS

LPDS

FCS

Control Ill.1 t-6.6 27.9+ 1.0 1244k 136 353+37 215+-34 + genistein 31.2It2.1 17.3+3 - . ._ 3 508 AI30 Residual activity (%) (28%) (62%) (41%) (61%) .~_-___ ---_p-_ The fibroblasts were seeded (day 0) at IO” cells per well (35 mm diameter). At day 2, the cells were washed with PBS and incubated in 10% FCS medium or 5% LPDS medium for 8 h. Genistein (40 pg/mL) or DMSO (6 pL/mL) were then added to cells and

figubations were extended for 16 h. The binding and metabolismof -- I-LDL were determined as described in Materials and nzethods. The results represent means +, SD of two independent experiments. Assays were carried out in triplicate for each experiment.

gen.:

-

-

+

Fig 4. Effect of genistein on LDL receptor gene transcription in AS49 ceils. AS40 cells were plated at 8 x I(Y’cells per 80 cm2 flask (similar density as in figures 2 and 3). After 2 days of growth. cells were washed with S. After that, they were incubated in 10% FCS medium t’or 24 h or in 5% LPDS mejdium fyq# h extended for 16 h with genistein (40 pg/mL) or DMSO (6 pL/mL). 20 ~_lgof extracted total RNA were hybridised with 50 x 10 cpm (’‘P) of LDL receptor and 36 B4 probes as described in Mutc~r-icr1.s u/rd tmhotls. Controls are on lanes 1. 2. 3 and 4 (lene 1, LDL receptor and 36 B4 probes with RNases: lane 2. LDL receptor and 36 B4 probes without RNases: lane 3. hybridisation positive control of 36 B4: lane 4, hybridisation positive control of LDL receptor. The LDL receptor mRNA Icvel was determined. as indicated in Murcrids ad n~fhnd.~. by scanning densitometry and expressed. after correction for constant 36B4 probe, as a ratio relative to the FCS lane (histogram).

likely that tumour cells need a very high level of cholesterol during their growth phase. A high requirement for cholesterol during the exponential growth phase was further supported by the higher efficiency of LDL receptors. Indeed, the LDL internalisation index (metabolism/binding ratio) was higher in growing cells than in confluent cells (fig I and table I). This cholesterol might be used for membrane synthesis and could be related to cell growth. Mazzone rt al [3OJ have suggested that the onset of growth in fibroblasts leads to a redistribution of free cholesterol from intracellular compartments to plasma membrane. A similar effect on cholesterol redistribution can be obtained by high density lipoproteins, subclass 3 [31] and we have shown that these lipoproteins are able to promote A549 cells proliferation which is concomitant with the specific phosphorylation of a 20 kDa protein [32]. At the stage of confluence, cells might store the excess cholesterol after esterification by ACAT. Therefore, the quantity of free cholesterol available to modulate the sterol-response element (SW) of the LDL receptor gene [33] can vary during cell growth. The rise of LDL receptor activity in A549 cells during growth phase might be related to a sterol-independent

mechanism. A stL’rol_indL’pc’nclcnl pathway has been shown in human liver cells [ 17, 18. 34. 351 and it has been suggested that this pathway could play a11 important role it1 r*ilw when cholesterol Icvels in the blood are high and the LD’L receptors are down-regulated [ 181. Cytokines and growth factors are able to significantly up-regulate LDL receptor expression [36-381. Growth factors acting through tyrosine kinase, such as insulin and platelet-derived growth factor, increase LDL receptor activity in fibroblasts and arterial smooth muscle cells 130, 391. The stimulation of tyrosine kinase activity induces the up-regulation of LDL receptor expression in HepG2 cells [ 191 and in smooth muscle cells [38J, as a consequence of a higher transcription and surface expression of the LDL receptor [38]. The autocrine growth factors loop in A549 cells could contribute Aothe higher expression level of the LDL receptor gene: Siegfried 1201 has shown that AS49 cells produce several growth factors with two of them known to stimulate tyrosine kinase activity (TGFcc and IGFl). Both cholesterol and genistein were shown, in our study. to be able to inhibit LDL receptor actikbty. However, cholesterol (from FCS) behaves as a powerful inhibitor in confluent cells in contrast to genistein that behaves as a better

880

inhibitor in exponentially growing cells. The 10~ efti:ct of cholesterol in growing cells seems to be a characteristic of tumour cells since it has been observed also in other malignant cells [40]. The low efficiency of genistein in confluent cells can be explained by a lower tyrosine kinase activity. Indeed, LDL binding to smooth muscle cells at near confluence has been shown to be unmodulated by genistein. However, if LDL binding is previously enhanced by a tyrosine kinase-stimulating growth factor (bFGF), genistein is able to inhibit LDL binding [38] The LDL receptor seems therefore to be regulated by two mechanisms in A549 cells. The tyrosine kinase-dependent mechanism is predominant during the exponential growth phase: genistein highly inhibits LDL receptor activity. Moreover, deprivation of cholesterol gives only a slight enhancement of the LDL receptor activity and did not induce any effect in the presence of genistein (table I). The steroldependent mechanism is predominant in cells at confluence: deprivation of cholesterol strongly enhanced LDL receptor activity and, contrary to growing cells, an enhancement was also observed in the presence of genistein. Both mechanisms also seem to be present in normal cells (fibroblasts) but, in contrast to tumour cells, the sterol dependence during the growth phase is much higher and can be observed even after inhibition of LDL receptor activity by genistein (table II). Tyrosine kinase activity seems therefore essential for allowing growing cells to obtain a high quantity of cholesterol that can be further used in membrane formation. The LDL receptor and HMG CoA reductase genes have a co-ordinated [ 11 and multilevel 12, 401 regulation. Transcriptional and post-transcriptional mechanisms (translation, protein degradation) have been clcmonstrated for MMG CoA reductase 121. A transcriptional regulation of the LDL receptor by cholesterol is well established and a post-RNA regulation has been suggesred 1401 since receptor activity increases more than mRNA level during cholesterol deprivation. The effect of genistein was accompanied, in our study, by a complete disappearance of specific mRNA. The band of the 36B4 mRNA probe had also a decreased intensity (fig 4) but it is likely the consequence of a lower deposition of materials. Indeed, a decrease in 36B4 gene transcription by tyrosine kinase inhibitors has never been reported to our knowledge and, moreover, it has been shown that the modulation of a tyrosine kinase receptor (IGF-I receptor) did not have any effect on the expression of 36B4 mRNA 1411. The up-regulation of LDL receptors by tyrosine kinase might be the consequence of a stabilisation of mRNA or a higher transcription of the LDL receptor gene. This last event might be mediated by the nuclear signal transducer Egr-1, previously described by Liu et a![ 191. The question arises as to the possible relationship between the high need for cholesterol in a growing neop tumour and the serum cholesterol level. A low serum level has been demonstrated at the time of cancer dkgnosis in a large number of epidemiological studies: it has been termed

‘tumour-associated hypocholesterolemia’ 1421. T cholesterolemia seems to be the direct consequence of the malignancy [43]. Taken together, our results suggest that ~11s have high adaptive capacities concerning exogenous cholesterol ply. The tyrosine kinase pathway allows cells to up-reg their LDL receptor activity when the sterol dependence is deficient (tumour cells) or when cells need a high quantity of cholesterol (normal exponentially growing cells and tumour cells).

We thank Dr John Woodward and Dr G Cockiblingum for critical reading of the manuscript. This research was supported by the FddCration Nationale des Centres de Lutte Contre le Cancer, the Cornit& Ddpartementaux of the Ligue Nationale contre le Cancer (r&ion Midi-Pyr&Ces), the Conseil Regional Midi-Pyre&es and the Minis&e de I’Enseignement SupCrieur et de la Recherche.

eferences I Goldstein JL. Brown MS (1984) Progress in understanding the LDL receptor and HMG CoA reductase; two membrane proteins that regulate the plasma cholesterol. J Lipid Res 25. 1450-1461 2 Goldstein JL. Brown MS ( 1990) Regulation of the mevalonate pathway. N~ctlcUJ 343 425-430 3 Ho YK. Gihrton G. Ost A, Peterson C ( 1978) Low density lipoprotein (LDL) receptor activity in acute myelogenous leukemia cells. Blood 52, 1099-l I I4 4 Vitols S, Gahrton G. Ost A, Peterson C (1984) Elevated low density lipoprotein receptor activity in leukemic cells with nwnocytic differcntiation. Nloo~lA3. I I X6-I IO.? 5 VitoI\ S, Peterwn c‘. L,itrhh(jtl0. I-hltn P, Ahcrg B ( 1992) Elevated uptake of low density lipoproteins by human lung cancer tissue irr viw. Cwx~r~ Res 52. 6244-6247 6 Gueddari N. Favre G, Hachcm H, Marek E, Lc Gaillard F. Soula G (1993) Evidence for up-regulated low density lipoprotein receptor in human lung adenocarcinoma cell line AS49. Bioc~hinric~ 75, 8 I l-8 19 7 Chait A, Bierman EL, Albers JJ (1979) Low density lipoprotein receptor activity in cultured human skin fibroblasts. J Clirr /tr\w/ 64, I3091319 8 Kruth HS, Avignan J, Gamble W, Vaugnan M (1979) Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts. ./ Cell Biol83,588-594 9 Stein 0. Stein Y ( 1975) Surface binding and interiorization of homologous and heterologous serum lipoproteins by rat aortic smooth muscle cells in culture. Biochin~ Biophy Am 398. 377-384 10 Vlodavsky I, Fielding PE, Fielding CJ, Gospodarowicz D ( 1978) Role of contact inhibition in the regulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells. P I’O~Nurl Ad Sci USA 75, 356-360 I I Kenagy R, Bierman EL, Schwartz S (1983) Regulation of low density lipoprotein metabolism by cell density and proliferative state. J Cell P Irvsiol I 16. 404-408 i2 Gal D, Mac Donald PC, Porter JC. Smith JW. Simpson ER ( 1981) Effect of cell density and confluency on cholesterol metabolism in cancer cells in monolayer culture. Cancer Res 41.473-477 I3 Auwerx JH, Chait A, Wolfbauer G, Deeb SS ( 1989) Involvement of second messengers in regulation of the low density lipoprotein receptor gene. Mol Cd Bid 9, 2298-2302

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