Defective Low-Density Lipoprotein Metabolism in Cultured, Normal, Transformed, and Malignant Keratinocytes

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1 22-22X/84/ $2./ THE J OU RNA L OF I NVESTtr.AT IVE D ER MATOLOGY, 83:436-44,! 984 Copyright 1984 by The Willia ms & Wilkins Co. Vol. 83, No.6 Printed in U.S.A. Defetive Low-Density Lipoprotein Metabolism in Cultured, Normal, Transformed, and Malignant Keratinoytes MARIA PONEC, PH.D., LOUIS HAVEKES, PH.D., JOHANNA KEMPENAAR, SJAN LAVRIJSEN, M.D., AND BERT JAN VERMEER, M.D. Departments of Dermatology, University Hospital and Gaubius Institute TNO (LH), Leiden, The Netherlands To obtain more information on differenes in ellular behavior during the differentiation proess, a number of types of epithelial ells with and without a defet in ornified envelope formation were ompared as to the regulation of intraellular holesterol synthesis by lowdensity lipoprotein (LDL) and the uptake and degradation of [ 125 l )LDL. The following ells were ultured: normal skin fibroblasts (F), normal (K) and SV 4 transformed (SVK1 4) keratinoytes, and a number of squamous arinoma ell (SCC) lines in whih the defetive terminal differentiation was found to our in the following order of intensity: SCC-12F2 < SCC-25 =o SCC- 15 < SCC-12B2 < SCC-4. Compared with normal human fibroblasts, most of the ells under study showed a defetive response to hanges of the extraellular serum LDL onentration. The degree of induibility of holesterol synthesis after the ells were deprived of extraellular soures of holesterol as well as the degree of the LDL-indued suppression of the intraellular holesterol synthesis in ells preinubated in medium supplemented with lipoproteindefiient serum dereased in the following order: F > SCC-4 > SCC-15 :o SCC-25 > SCC-12B2::::: SCC-12F2 > SVKH E K. A defet in LDL metabolism was found to be responsible for the partial or omplete failure of LDL to regulate the holesterol metabolism, beause when sterol was delivered to all ell types in artifiial nonlipoprotein form (i.e., as 25-hydroxyholesterol) a marked suppression of holesterol synthesis was observed. For all SCC lines tested exept SCC-12B2 good orrelation was found between the degree of LDL-indued suppression of holesterol synthesis and the dereasing ability of ells to differentiate into squames or ornified envelope-forming ells. The transformation of keratinoytes by SV 4 virus did not lead to any hange in the response of the ells to hanges in the extraellular LDL onentration sine both the normal and the transformed keratinoytes showed the same response to LDL (i.e., no response). Manusript reeived Deember 8, 1983 ; aepted for publiation June 18, Reprint requests to: Dr. Maria Pone, Department of Dermatology, Unive rsity Hospital, Rijnsburgerweg 1, 2333 AA Leiden, The Netherlands. Abbreviations: DDT: dithiothreitol DV-H: Dulbeo-Vogt's a nd Ham's 12 medi a FCS: fetal alf serum NBCS: newborn alf serum LPDS: lipoprotein-defi ient newborn alf serum LDL: low-density lipoprotein SCC: squamous e ll arinoma SDS: sodium dodeyl sulfate SVK,.: keratinoytes t ransformed by SV 4 virus HMGCoA: 3-hydroxy-3-methylglutaryl oe nzyme A Human fibroblasts and many other types of ultured ells supply themselves with holesterol originating from low -density lipoproteins (LDL) whih the ells ta ke up by a reeptormediated mehanism [1,2]. Aording to this mehanism, the LDL is first bound to a speifi plasma membrane reeptor, and then internalized and degraded in lysosomes. The holesterol liberated from LDL during its degradation has been proved to regulate the intraellular holesterol synthesis by inf1uening the ativity of 3-hydroxy-3-methylglutaryl oenzyme A (HMGCoA) redutase. We reently showed [3] that human epidermal keratinoytes differ from most extrahepati ells in the regulation of holesterol synthesis; a defet in LDL metabolism was found to be responsible for the failure of LDL to regulate holesterol synthesis in these ells. It an be expeted that aner ells, whih repliate ontinuously, require large amounts of holesterol for the synthesis of ell membranes. Suh holesterol ould be supplied by de novo holesterol synthesis or by assimilation of holesterol from plasma lipoproteins. Reent studies have shown differenes in the response of various types of aner ells to LDL. On the one hand, Gal eta! [ 4) found that a number of types of aner ells are able to bind and degrade LDL to a higher degree than non-neoplasti ells an. On the other hand, Gal et al (5) and Anderson et al [6] found that some ell lines (epidermoid ervial aner ell line EC-168 or human epitheloid arinoma A-431 ell s) were able to bind LDL to a high degree but had defetive internalization of LDL. Both of the latter ell types are of epithelial origin and are differentiating ells. In stratified squamous epithelia, a balane between ell division and terminal differentiation assures the maintan'=! of tissue size. In malignant ells this balane is disturbed. Sine normal keratinoytes failed to respond to LDL and some malignant ells of epithelial origin showed a defetive response to LDL, the questions arose as to whether there is any orrelation between the ability of ells to differentiate to ornified ells and the degree of the regulatory effets of LDL on intraellular holesterol synthesis. Rheinwald and Bekett [7,8] reently sueeded in ulturing and haraterizing the growth properties of a number of ell lines derived from human squamous arinomas (SCC lines), and found that in these ells the mehanism that triggers terminal differentiation was defetive to various degrees as indiated by the finding that se exhibited a redued rate and degree of ornified envelope formation when inubated in a semisolid medium. Taylor-Papadimitriou et al (9] and Steinberg and Defendi [1,11] showed that ultured keratinoytes transformed by SV 4 virus (SVK" ells) showed, like the SCC ells, impaired ability to differentiate into ornified ells, and the SVK,. ells differ from the normal ells from whih they are derived by an absene of dependene on 3T 3 feeder layer. The aim of the present investigation was to determine whether a defet in the terminal differentiation program affets the regulatory mehanism for the holesterol synthesis by extraellular LDL. For these studies, a number of SCC lines and normal and SV 4-transformed keratinoytes were used. 436

2 De Cell Culture MATERIALS AND METHODS Fibroblasts: The isolation a nd ulture of human skin fibroblasts were performed as desribed elsewhere [12]. For the experiments, 1.3 X 1 4 / m 2 ells were inoulated in Petri dishes (G reiner) ontaining Ham's F 1 medium supplemented with 15% newborn alf serum (NBCS) and a mixture of ant ibiotis (streptomyin 1 mg/ml and peniillin 1 U/ml), and inubated in a humidified atmosphere with 5% C 2. The medium was renewed twie a week. The ultures reahed onflueny in abou t 1 week. The 5th to loth passages were used for the experiments. Keratinoytes: The keratinoytes ori ginating from juvenile foreskin wer e isolated and ul tured aording to a slightly modified version of the method of Rheinwald and Green [1 3], the details of whih have been desribed elsewhere [14]. For the experiments, 2 X JO'/m 2 keratinoytes were plated together with 4 x 1'/m 2 feeder ells (gammairradiated [3 R] 3T3 mouse fibroblasts) on Petri dishes in a mixture of Dulbeo-Vogt's and Ham's F 12 media (3 :1 ) (DV-H medium) supplemented with 5% fetal alf serum (FCS), a mixture of antibiotis, 1-6 M isoproterenol, and.4 11g hydroortisone per mi. After 3-4 days, the medium was replaed by a medium of the same omposition but also ontaining 1 ng epidermal growth fator per ml (Sohiba, Frane). This medium was renewed twie a week. The ultures were onfluent within 1-14 days after plating. The l st to 3rd passages were used for the experiments. S VK 14 ells: SV 4-transfonned normal human epidermal keratinoytes were kindly provided by Dr. J. Taylor-Papadimitriou; 1.5 x 1 4 ells/em"' were plated on Petri dishes ontaining DV -H medium supplemented with 5% FCS, antibiotis,.4 11g/ml hydroortisone, and 1-6 M isoproterenol. SV l\: 1 1 e ll were grown in the absene of fibroblast feeder support. The medium was renewed twie a week. The ult ures reahed onflueny in 5-8 days. The 17th to 42nd passages were u sed for the experiments. SCC lines: Human squamous arinoma ell lines SCC-12F2 and SCC-l2B2 (originating from faia l epidermis) and SCC-4, -15, and -25 (originating from the tongue) were kindly provided by Dr. J. Rheinwald; 1.5 X 1"' e lls/em' were plated together wi th 4 X 1' lethally irradiated 3T3 ells/em' in Petri dishes ontaining DV-H medium supplemented wit h 5 % FCS, antibiotis, and.4 11g/ ml hydroortisone. The medium was renewed twie a week. The ultures reahed onflueny in 8-1 days. C ult ures of keratinoytes and SVK 1, and SCC ells were kept at 37"C in a humidified atmosphere ontaining 1% CO, and 9% air. One day before the experiments were started the e lls were fed with DV-H medium upplemented only with 5% FCS and antibiotis. Lipoproteins: Human LDL (density = g/ml) was isolated fro m plasma of hea lthy subjets by density gradient ultraentrifugation (15] followed by tube sliing. Lipoprotein-defiient newborn alf serum (LPDS) (density> g/ml) was prepared by ultraentrifugation at a density of g/ml followed by dialysis aga inst saline. LDL was labeled with 12f J aording to Bilheimer eta! [ 16]. Assays Assessment of the inorporation of /"C}aetale inta f'c)holesterol: For onditioning, the ells were inubated for 24 h with Ham's F 1 (fibroblasts) or Dulbeo-Vogt medium (other types of ell), both on taining either 15% NBCS or 15% LPDS. To assess the effets of LDL or extraellular holesterol on the de novo holesterol synthesis, the ells were preonditioned in medium ontai ning LPDS and reinubated for 6 h in medium supplemented with LDL or 25- hydroxyholesterol (Steraloids) at onentrations of g holesterol/ ml medium. 25-Hydroxyholesterol was added in 5 11! ethanol/dish. The ontrols reeived on ly 5 d ethanol. For the measurement of de novo holesterol synthesis, [ 14 C]aetate (The Radiohemial Centre, Amersham, 59 mci/ mmol ; Ci/m l medium ) at a final onentration of.5 rom was added just before the e lls were inubated for 18 h. All inubations were performed at 37"C in a humidified atmosphere ontaining 5 % C 2 (for fibroblasts) or 1% C 2 (for other e lls). After inubation the medium was removed and ells were washed 3 times with phosphate-buffered saline and digested in 1 N NaOH. A sample was taken for protein determination [17] and t he amount of [ 14 C] holesterol was measured in t.he ell fration and medium aording to Goldstein et al [18] with a slight modifiation. The extrated lipids were separated by t hin -layer hromatography, using Kieselgel 6 plates (Merk) in sequent ial elution, i.e., hloroform:methanol (98:2) followed by holoroform:n-hexane (25:6). In the first system about two-thirds LDL METABOLISM IN KERATINOCYTES 437 of t he plate was developed. After t he seond elution the R 1 value for holesterol was.3. For all samples, a orretion was made fo r reovery by adding an internal standard of [1.3-3 H(N)]holesterol (New England Nulear, 53 Ci/ mmol) at the beginning of t he extration proedure (3 X 1" 1 dpm/sample). Reovery ranged between 65-8%. Cell Assoiation. (Binding Plus Internalization) and Degradation. of 1 "'1- Labeled LDL Before t he assay, t he ells were preinubated with LPDS-ontaining medium to ex press the LDL reeptor. [ 125 l] LDL was then added (-5 11g/mll and the ells were reinubated at 37 for 3 h. For the assessment of nonspeifi binding, 2 11g of unlabeled LDL/ ml was added to the medium ontaining [ 125 I]LDL. The ell assoiation and degradation of [ 125 l)ldl were measured essentially aording to Goldstein et al [19]. In short, degradat ion was measured as free-iodine trihloroaeti aid-soluble radioativity in the medium. After washing the ells with albumin-ontaining buffer, exatly as desribed by Goldstein et al [1 9], the e lls were dissolved in.2 N NaOH before oun ting for radioativity and protein determination [1 7]. Cross-linked Envelope Formation The ross-linked envelopes formed eit her spontaneously or after ionophore treatment in onfluent ultures were determined aording to Ri e and Green [2). Briefly, the e lls harvested by trypsinization were divided into two frations. The first fration was immediately treated with sodium dodeyl sulfate (SDS, 1 %) and dithiothreitol (OTT, 2 mm) for 5 min at 1o. The seond fration was resuspended in serum-free medium ontaining ionophore X 537 A at a onentration 5 11g/ml (Sigma) and inubated for 2 h at 37. The ells were t hen boiled for 5 min in the presene of SDS and DTT. The envelopes were sored by phase-ontrast mirosopy. RESULTS The rate of the total de novo holesterol synthesis (measured as the inorporat ion of [ 1 " 1 C]aetate into [ 14 C]holesterol during a period of 18 h a nd expressed per mg of ellular protein) was found to differ among the various types of ells under study (Table I). After inubation in m edium supplemented with NBCS, the rate of holesterol synthesis was high in both the normal and sv 4-transformed keratinoytes, lower in se ells, and lowest in fibroblasts (Table I). Removal of exogenous holesterol (by inubation in medium supplemented with LPDS) had no effet on de novo holesterol synthesis by eit her the normal or t he transformed keratinoytes TABLE I. Comparison. of the de novo holesterol synthesis in. various onfluent ell ultures Cholesterol Pere nt holesterol synthesized" exret.ed into the in t.he pre!'.ne of: mediun'l ell in the presene of: 15% 15% 15% 15% NBCS LPDS NBCS LPDS Rntio LPDS/ NBCS Fibroblast SCC SCC SCC SCC-12B SCC- J2F a 2.2 SVKI, a Keratinoyte The onfluent e ll ultures were preinubated for 24 h in medium supplemented with either 15% NBCS or 15% LPDS. For the measurement of the de novo holesterol synthesis [ 14 Cjaetate ( Ci/ ml medium;.5 mm) was added and the ells were further inubated for 18 h. Subsequently, [ 1 "'C]holesterol was measured in both e ll layer and medium. " nmol of inorporated aetate/ mg protein. b Ratio of t he amount of holesterol synthesized by ells inubated in medium supplemented with lipoprotein-defiient newborn alf serum (LPDS) to that synthesized by e lls inubated in medium supplemented with newborn a lf serum (NBCS).

3 438 PONEC ET AL Vol. 83, No.6 -a... N d... >-. L LJ.. u u... - "-----::::..-==--=::=:!--==._=:==_::=_:=--==--=:...=- S ( ( -12 F 2 SCC-1282 SC C-25 s (( <> o $(( F 5 }Jg LDL/ml FIG 1. The effet of LDL on the inorporation of l "C]aetate into [ "C]holesterol. The on!luent e ll ultures were preonditioned for 24 h in medium supplemented with 15% LPDS and subsequently inubated for next 6 h in medium of the same omposition but in the presene of inreasing amounts of LDL (- 5 JLg protein/ ml), after whih ["C] aetate (Amersham, 59 mci/ mmol, 2.5 JlCi/ml medium,.5 mm) was added. After the overnight inubation the synthesized I' ''Cjholesterol was measured in the medium and the e ll separately. The values given in the figure represent the sum of l "C]holesterol present in the e lls and medium and are the means of dupliate determinations. K (keratinoyte) (X), SVK 14 ( ), SCC-12F2 (), SCC-1282 (.), SCC-25 ('17), SCC-15 (e), SCC-4 (), and F (fibroblasts (). TABLE II. Suppression of the de novo holesterol synthesis Cell Fibroblast SCC-4 SCC-15 SCC-25 SCC-1282 SCC-12F2 SVK 14 Keratinoyte by 25-hydroxyholesterol Perent of ontrol ino rporation of I u ]aetate into holesterol (l'g 25- hydroxyholesterol added per ml medium) The ells we re inubated for 24 h in medium supplemented with 15% LPDS. Next, the medium was renewed and either no or 1-5 Jig of 25- hydroxyholesterol was added per ml medium and the e lls were inubated for 6 h. ["C]Aetate (2.5 JlCi/ ml medium;.5 mm) was then added and the e lls were inubated for 18 h. Subsequently, the inorporation of [' ''C]holesterol was measured in both e ll layer and medium. but led to a n inreased holesterol synthesis in all se lines (Table I). The a mount of the de novo-synthesized holesterol exreted into t he medium was low for normal and transformed keratinoytes (less than 5%), somewhat higher for se ells (8-15%), and high for fibroblasts (5-6%). For all ells, the amount of holesterol exreted into t he medium was lower after inubation in medium ontaining LPDS. The addition of LDL to ells preinubated in medium supplemented with LPDS did not affet the holesterol syn thesis in either normal or t ran sformed keratinoytes but led to suppression of holesterol synthesis in all se under study. T he normal skin fibroblasts showed t he highest degree of LDLindued suppression of holesterol syn thesis (Fig 1). The addition of low onentrations of 25-hydroxyholesterol (5 11g/ml) led, in a ll of the ells under study, to a marked suppression of the de novo holesterol synthesis (Table II). The degree of LDL-indued suppression of holesterol synt hesis was independent of ell density for both SCC-12F2 and SCC-25 ells, whereas the derease of the total holesterol synthesis with inreasing ell density was more pronouned in SCC-25 than in SCC-12F2 ells (Fig 2). In the experiments in whih the metabolism of [' 5 I]LDL was followed, normal and transformed keratinoytes a nd SCC- 12F2 ells showed a low degree of ell assoiation and degradation of [ ' "I]LDL, SCC-25 ells showed a higher degree, and fibroblasts were the most ative in this respet (Fig 3) ac w. N..._ "f---o......_.. L... >-. 1/1 u 6 - '-- '- 1/ u... u... 2 ' -a <lj N 2 "' e... >-. 1/1 --- e \ ""-.. <lj 1 mg protein/ dish 1/1 <lj Qj... u "'.. 1 E E mg protein /dish FIG 2. The effet of e ll density on the de novo holesterol synthesis in SCC-12F2 and SCC-25 e lls. The ells were inoulated at various densities ( X 1 6 ells/go mm Petri dish) and ultured for 2 days. The ells were then washed with PBS and preonditioned for 24 h in medium supplemented witb 15% LPDS. The ells were subsequently inubated for 6 h in medium of the same omposition either in the absene (a) or presene of LDL (1 Jlg/ml) (b). Thereafter ["C] aetate (5 llci/ dish) was added and after the overnight inubation the synthesized ["C]holesterol was analyzed. The values given in the figure are the mean of tripliate determinations. Perent of ontrol inorporation: inorporation of the ["C]aetate into holesterol (expressed as dpm/mg protein) in the presene of 1 Jig LDL/ ml wa related to the inorporation in the ontrol djshes. SCC-12F (), SCC- 25 (e).

4 De "'1 C1J a.,ooo L.. C1J. en E.. en --' Cl --',1 I I Q 2 5 SCC- 25 Binding+ Internalization SCC-12F2 SVK14 K 2 p p v j Degradation v 1 fl I- LDL )Jg/ml FIG 3. The ell assoiation (binding plus internalization) and deg radation of ( 125 l]ldl by various ultured ells. After 24 h preinubation in medium supplemented with 15% LPDS the onfluent ell ultures were further inubated for 3 h at 37 in the presene of inreasing amounts of ( 125 l]ldl (-5 11 g/ ml). The degradation of [ 125 l]ldl was measured as iodine-free trihloroaeti aid-soluble radioativity in the medium. The binding plus internalization of [ 125 I]LDL was taken as the total amount of ell-assoiated radioativity. Eah value is the mean of dupliate determinations. -, Total ell assoiation or degradation; -. nonspeifi ell assoiation or degradation measured in the presene of 2 ll.g/ ml unlabeled LDL; -, speifi ell assoiation or degradation as alulated by subtration of nonspeifi ell assoiation or degradation from, respetively, total ell assoiation or degradation. The experiments on ross-linked envelope formation revealed that in onfluent ultures the level of spontaneous envelope formation was very low in SVK,, ells and in most of the SCC lines and higher in normal keratinoytes. The ionophore treatment led to a marked inrease of the level of ross linked envelope formation in most of the ell types under study. Of the normal and virally transformed keratinoytes, 9-1% had the apaity to form ornified envelopes. In SCC lines the ompeteny to form ornified envelopes was lower, ranging between 8-65% (Table III). DISCUSSION The present results show that ompared with human skin fibroblasts, most ofthe types of ells under study had a defetive LDL METABOLISM IN KERATINOCYTES 439 TABLE III. Formation of ross-linked envelopes by various ells Cell Cross-linked envelopes formed(%) Spontaneously After ionophore treatment Keratinoyte SV K SCC-4 <3 8-9 SCC-12B2 < SCC SCC-12F The onfluent ell ultures were trypsinized and the ells were split into two fration s. The first fration was immediately treated with SDS and DTT. The seond fration was resuspended in serum-free medium ontaining ionophore and inubated for 2 h at 37. The ells were then treated with SDS and DTT. The envelopes were sored by phase-ontrast mirosopy and the results are expressed as perentage of total ell number. response to hanges in the extraellular LDL onentration. Among these ells, both the induibility of holesterol synthesis by inubation in medium supplemented with LPDS instead of NBCS and the LDL-indued suppression of holesterol synthesis in ells preonditioned in LPDS-ontaining medium showed the following order of intensity: fibroblasts > SCC-4 > SCC- 15 = SCC-25 > SCC-12B2 SCC-12F2 > SVK 14 normal keratinoytes (Table I, Fig 1). However, in all of these ell types the addition of low onentrations (1-5 1!g/ml medium) of 25-hydroxyholesterol, whih enters ells easily, led to more than 9% suppression of holesterol synthesis (Table II), whih demonstrates the presene of a mehanism ontrolling the ativity of HMGCoA redutase. Compared with that of fibroblasts, the metabolism of [' 25 1] LDL was impaired in SCC-25 ells and virtually absent in SCC- 12F2 and SVK" ells and normal keratinoytes (Fig 3). This means that a defet in the metabolism of LDL was responsible for the partial or omplete failure of LDL to regulate holesterol metabolism in these ells. A similar effet has been seen in human epitheloid arinoma A-431 ells [6] and the epidermoid ervial aner ell line EC-168 [5). The surfae of the latter ells was found to possess a large number of LDL reeptors. However. only a _small fratio of the LDL-reeptor omplexes was assoiated with oated regwns and internalized by the ells. In our investigations both the normal and the SV 4-transformed keratinoytes showed virtually no binding of LDL to the. memb:ane surfae in either the biohemial or the morphologi. studies. The results of the latter (not shown) indiated that m some of the SCC ells LDL was abundantly distributed in nonoated regions on the ell surfae and that only a small perentage had been internalized (B. J. Vermeer eta! manusript in preparation). ' Compared with normal and transformed keratinoytes all of the se lines under study showed a lower rate of holsterol synthesis (Tab! 1). This finding was quite surprising, beause aner ells rephate at a high rate in vivo and therefore ould be expeted to need a greater amount of holesterol for mem brane snthesis. In all SCC lines the regulatory mehanism underlymg holesterol metabolism by serum lipoproteins was found to be operative to a ertain degree. For most of t hese ells, the intensity of LDL-indued suppression of holesterol synthesis orrelated with defetiveness of the terminal differetiation program [7] (Table III). The only exeption was seen with the SCC-12B2 line whih with t he SCC-12F2 line are subopulatios of the SCC-12 line derived from the squamous armoma of the heek. In both of these ell lines the LDLindued suppression of holesterol synthesis was of the same order of very low intensity (Fig 1). However, ompared with the well-differentiating SCC-12F2line, the SCC-12B2 line had a long survival half-time in semisolid medium and showed a low degree of ornified envelope induibility [21] (Table III).

5 44 PONEC ET AL On the other hand, in SCC lines originating from the tongue, the degree of impairment of the ability to differentiate into ornified ells was orrelated with the degree of LDL-indued suppression of holesterol synthesis. It is oneivable that besides the defet in terminal differentiation of the epithelial tumor ells, the primary tumor site plays an important role in the mehanism for the regulation of holesterol synthesis by extraellular LDL. A number of studies have shown that holesterol metabolism in ultured ells is a funtion of ell density [22-24], i.e., the rate of proliferation dereases with inreasing ell density, and less holesterol is needed for t he assemblage of new membranes. We reently showed [3) that keratinoytes behave differently in this respet: t he rate of holesterol synthesis did not hange with inreasing ell density. In the present study similar results were obtained with well-differentiating SCC-12F2 ells, where an inrease of ell density had very little effet on holesterol metabolism (Fig 2). In less differentiating SCC-25 ells, inreasing ell density led to diminished holesterol synthesis, as found earlier for fibroblasts [3]. In both SCC-12F2 and SCC- 25 ells the LDL-indued suppression was independent of ell density (Fig 2). As shown by Rie and Green [2], a distintive feature of the terminal differentiation proess is the synthesis of ross-linked protein envelopes at the ell periphery. These authors found that only a small proportion of ultured normal keratinoytes form envelopes spontaneously, but a majority have all the required ingredients. When t reated with suh agents as ionophores, whih permit a flux of alium into ytoplasm whih ativates the ellular transglutaminase, the envelopes beome ross-linked. The SVK1 ells were shown by Taylor-Papadimitriou et al [9], and also were seen by us, to have an impaired ability to differentiate into ornified ells when the number of ells that had spontaneously formed ornified envelopes was taken as the measure of defetiveness. Unlike Taylor-Papadimitriou et al [9], we found that upon ionophore treatment the SVK14 ells were apable of forming ross-linked envelopes to the same degree as normal keratinoytes, whih suggests that these ells possess the omponents required for envelope assembly. This disrepany onerning the ompeteny of SVK 14 ells for ross-linked envelope formation may have been due to differenes in ulture onditions. Sine the normal and virally transformed keratinoytes showed the same degree of ornified envelope induibility, it is not surprising that both ell types behave similarly with respet to the regulation of holesterol synthesis. Namely, SV 4-transformed kerat inoytes had a high rate of de novo holesterol synthesis, whih was found not to be ontrolled by serum lipoproteins. This means that the normal extraellular ontrol of intraellular holesterol synthesis was absent in both types of ell. Our results suggest that the transformation of normal keratinoytes by SV 4 virus does not lead to a hange in the regulation of holesterol synthesis by lipoproteins. Similar observations have been made by Wu et al [25], who found that in SV 4-transformed human lung fibroblasts the metabolism of LDL proeeded in the same way as in normal fibroblasts. Sine the regulation of holesterol synt hesis seems to be unaltered in a loned ell line of keratinoytes transformed by SV 4 virus, these ells (whih showed unlimited growth potential and proliferate in the absene of fibroblast feeder) an serve as a model in studies on the mehanism underlying the regulation of holesterol synthesis in human keratinoytes. We would like to thank Dr. J. 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