high-density lipoproteins and the proliferation of human ......ing the growth of various cell types...

[CANCER RESEARCH 42, 3704-3713. September 1982]0008-5472/82/0042-OOOOS02.00

High-Density Lipoproteins and the Proliferation of Human Tumor Cells

Maintained on Extracellular Matrix-coated Dishes and Exposedto Defined Medium1

D. Gospodarowicz, G.-M. Lui, and R. Gonzalez

Cancer Research Institute and the Department of Medicine, University of California Medical Center, San Francisco, California 94143

ABSTRACT

The ability of high-density lipoprotein (HDL) to support the

growth of an established tumor cell line exposed to definedmedium supplemented with transferrin has been examined.Low-density A-431 carcinoma cells maintained on extracellularmatrix- or fibronectin-coated dishes proliferated actively when

exposed to a synthetic medium supplemented with HDL, 500fig protein per ml. Epidermal growth factor added at concentrations above 0.5 ng/ml inhibited cell growth, while at concentrations above 5 ng/ml it was cytotoxic. Among the varioussubstrata tested for their ability to support the active proliferation of low-density A-431 cells when exposed to transferrinand HDL, plastic was the least efficient. On fibronectin-coated

dishes, cells ceased to proliferate after 8 population doublings,while on extracellular matrix-coated dishes cells could be pas

saged for 50 population doublings. In the case of colon carcinoma, rhabdomyosarcoma, and Ewing's sarcoma cells ex

posed to medium supplemented with transferrin, the additionto the cultures of HDL alone resulted in a growth rate and finalcell density which were similar to those observed when cellswere exposed to serum-supplemented medium. In the case ofthe mammary carcinoma cell lines MCF-7 and ZR-75-1, HDL

also supported cell growth, although to a lesser extent than didserum. The present study therefore indicates that HDL is capable of supporting, either totally or partially, the in vitroproliferation of tumor cells.

INTRODUCTION

HDL2 in combination with transferrin have been reported to

support the in vitro proliferation of a number of normal diploidcells exposed to a synthetic medium and maintained on dishescoated with an ECM produced by cultured bovine cornealendothelial cells (14, 19, 20, 37). While vascular endothelialcells require only HDL and transferrin in order to proliferate atan optimal rate (37), both vascular smooth muscle cells (20)and corneal endothelial cells (14) require, in addition to HDLand transferrin, the presence of somatomedin C and EGF inorder to proliferate actively.

The ability of lipoproteins such as HDL to support the proliferation of tumor cells has not yet been investigated. However,based on their effect on the proliferation of normal diploid cells,the suspicion that they play an important role in the proliferationof tumor cells and possibly in the neoplastic process in vivo isreasonable. It is equally possible that during the transformation

' This work was supported by Grants EY 02186 and HL 23678 from NIH.2 The abbreviations used are: HDL, high-density lipoproteins; ECM, extracel

lular matrix; EGF. epidermal growth factor; DME, Dulbecco's modified Eagle's

medium.Received November 6, 1981; accepted June 7, 1982.

process cells cease to require HDL in order to proliferateactively and would no longer be sensitive to that agent.

In order to define the growth requirements of tumor cells forHDL, we have analyzed their growth-promoting effect on various human tumor cell lines maintained under serum-free conditions. These cell lines are the A-431 carcinoma, Ewing's

sarcoma, a colon carcinoma cell line, a rhabdomyosarcoma,and 2 mammary carcinoma cell lines (MCF-7 and ZR-75-1).

In all cases, we have observed that HDL is able to promotethe proliferation of these various cell lines when they aremaintained under serum-free conditions.

MATERIALS AND METHODS

Materials. EGF was purified as described by Savage and Cohen(35). Fibronectin was purified from bovine plasma as described byEngvall et al. (11). When analyzed by slab gehpolyacrylamide gelelectrophoresis under reduced conditions, the purified fibronectin ranas a doublet with a molecular weight in the range of 220,000. Crystalline bovine serum albumin was obtained from Schwarz/Mann (Orange-

burg, N. J.). Insulin and transferrin were obtained from Sigma ChemicalCo. (St. Louis, MO.). DME, F-12 medium, and Roswell Park Memorial

Institute Medium 1640 were obtained from Grand Island Biological Co.(Grand Island, N. Y.). Calf serum and fetal calf serum were obtainedfrom Irvine Serum Co. (Irvine, Calif.). Tissue culture dishes were fromFalcon Plastics, gentamicin was from Schering Co. (Kenilworth, N. J.),and Fungizone was from Squibb (Princeton, N. J.).

Preparation of HDL. Human HDL (1.07 < d < 1.21 g/cu cm) wasobtained from human plasma by differential ultracentrifugai flotation(26). In order to further remove contaminating plasma proteins, theHDL preparations were washed by recentrifugation in solutions with adensity of 1.210 g/cu cm. Protein concentrations were determined asdescribed by Lowry et al. (29) and modified by Maxwell ef a/. (30).

The degree of low-density lipoprotein contamination in the purified

HDL preparations was analyzed by double immunodiffusion (37) andwas found to be less than 0.2%. To eliminate the possibility of acontamination by plasma proteins, the purity of the HDL preparationswas analyzed by slab gel electrophoresis (5 to 18%, exponentialpolyacrylamide gel gradient containing 0.1% sodium dodecyl sulfate)with or without prior delipidation with tetramethylurea (37). When theelectrophoretic patterns of HDL preparations were compared to that ofplasma, no obvious contamination by plasma proteins was observable.

Cell Culture Conditions. Cultures of bovine corneal endothelial cellswere established from steer eyes as already described (18, 22). Stockcultures were maintained on tissue culture dishes in DME supplemented with 10% fetal calf serum, 5% calf serum, gentamicin (50 jug/ml), and Fungizone (2.5 /ig/ml). Prior to being used, all media werepassed through a Millipore filter (0.2 firn). Fibroblast Growth Factor(100 ng/ml) was added every other day until the cells were nearlyconfluent.

A-431 carcinoma cells were obtained from 2 distinct sources. One

culture was obtained through the courtesy of the Naval BiosciencesLaboratory (University of California, Berkeley, Calif.) and was called A-431-NBL, while the other culture was obtained through the courtesy of

3704 CANCER RESEARCH VOL. 42

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HDL and the Proliferation of Tumor Cells

Dr. F. Fox (University of California, Los Angeles, Calif.) and was calledA-431-FF. Both cultures were propagated on tissue culture dishes and

grown in the presence of DME supplemented with 10% fetal calf serum,gentamicin (50 /uQ/ml), and Fungizone (2.5 Â¿ig/ml).

The colon carcinoma (HS-703T), human breast carcinoma (MCF-7),and rhabdomyosarcoma (A-204) cell lines were obtained from the

Naval Biosciences Laboratory (University of California, Berkeley,Calif.). The human breast carcinoma (ZR-75-1 ) cell line was obtained

through the courtesy of Dr. R. Stern (Department of Pathology, University of California, San Francisco, Calif.). Cultures of colon carcinomaand rhabdomyosarcoma were propagated on tissue culture dishes andgrown in DME supplemented with 10% fetal calf serum, gentamicin (50/Â¿g/ml), and Fungizone (2.5 Â¡ig/mV).Cultures of human breast carcinoma MCF-7 were grown in DME:F-12 medium mixed at a ratio of 1:1

and supplemented with 10% fetal calf serum, gentamicin (50/Kj/ml),and Fungizone (2.5 Â¿ig/ml). Cultures of human breast carcinoma ZR-75-1 were grown in Roswell Park Memorial Institute Medium 1640

supplemented with 10% fetal calf serum, gentamicin (50 fig/ml), andFungizone (2.5 fig/ml).

Preparation of Fibronectin- or ECM-coated Dishes. Fibronectin-

coated dishes were prepared as described by Kramer ef al. (27).Plastic dishes coated with an ECM produced by corneal endothelialcells were prepared using either detergent treatment (0.5% Triton inphosphate-buffered saline) as already described (17, 19, 21, 39) or

base treatment (NH<OH, 0.02 M in distilled water) (20). When basetreatment was used, confluent corneal endothelial cell cultures werefirst washed with distilled water and then exposed to 0.02 M NH4OH indistilled water for 5 min followed by washing with phosphate-buffered

saline (20). ECMs treated both ways were equally capable of supporting the growth of various cell types seeded on them (20).

Cell Seeding. Cells were detached from stock plates by exposingthem to a solution containing 0.9% NaCI, 0.01 M sodium phosphate(pH 7.4), 0.05% trypsin, and 0.02% EDTA (STV solution; Difco). Whencells rounded up, they were resuspended in DME supplemented with500 Â¡igprotein per ml HDL. The cell suspension was then spun down,and the cell pellet was resuspended in 2 ml of DME supplemented with500 /jg protein per ml HDL. When seeding was performed in thepresence of serum, the cell pellet was resuspended in DME containing10% fetal calf serum. An aliquot of the cell suspension was thencounted in a Coulter Counter or in a hemocytometer, and cells weredistributed at an initial cell density of 2 or 4 x 10" cells per 35-mm

dish as described below.Cell Growth Measurement and Culture Lifetime Determination.

For cell growth measurements and culture lifetime determinations, cellswere seeded at an initial density of 2 or 4 x 10" cells/35-mm ECM-

coated or fibronectin-coated dish. When cultures were to be exposedto serum-free medium, seeding of the various cell types was done in

their respective media supplemented with the various plasma factorsbeing analyzed (transferrin, HDL, or insulin present alone or in combinations and at the concentrations indicated in the figure legends). HDLand transferrin were added only once (Day 0), while insulin was addedevery fourth day. When cultures were to be maintained in the presenceof serum, cells were seeded in medium supplemented with 10% fetalcalf serum. Triplicate plates were trypsinized and counted with aCoulter Counter or in a hemocytometer, at appropriate times. Themorphological appearance of the cultures was analyzed by phase-

contrast microscopy, and pictures were taken once cultures were tobe terminated. Culture lifetime determinations were performed as already described (20, 21).

RESULTS

Effect of Substrate on the Proliferative Response of Low-density A-431 Carcinoma Cells Exposed to a Synthetic Me

dium Supplemented with Serum or with Transferrin, HDL,and Insulin Added Either Singly or in Combination. To analyze the proliferative response of A-431 carcinoma cells as a

function of the substrate upon which cells were maintained,low-density (2 x 10") A-431-NBL cells were seeded on ECM-

coated, fibronectin-coated, or plastic dishes. Cultures were

then exposed to the transferrin, insulin, and HDL added eithersingly or in combination. The density of such cultures after 8days was compared to that of cultures exposed to optimalconcentration (10%) of fetal calf serum. As shown in Chart 1,A-431-NBL cells maintained on ECM-coated dishes and ex

posed to DME alone did not proliferate appreciably over thatperiod of time (Chart 1X\). The addition of transferrin (10 fig/ml) or insulin (2.5 /tg/ml) either alone or in combination did nothave any significant effect. In contrast, the addition of HDL(500 fig protein per ml) made cells proliferate as well as whenthey were exposed to optimal (10%) serum concentration(Chart 1/0. The addition of transferrin (10 jug/ml) together withHDL had a small synergistic effect, while that of insulin resultedin a marked decrease in the final cell density (Chart 1X\). Thisinsulin effect could be observed at concentrations ranging from100 ng/ml to 10 /Â¿g/ml.3When the final cell density of A-431 -

NBL cells maintained on fibronectin-coated dishes (Chart 16)

and exposed to the various factors was compared to that ofcultures maintained on ECM-coated dishes, their final cell

density was the same regardless of the combination of factorsto which cells were exposed. Transferrin had a greater synergistic effect when added together with HDL, and insulin had asmaller inhibitory effect than that observed with cultures maintained on ECM-coated dishes. In both cases (either ECM- orfibronectin-coated dishes), the final cell density of cultures

exposed to HDL and transferrin was higher than or equal tothat of cultures exposed to 10% fetal calf serum. This indicatesthat HDL and transferrin can fully replace serum. When cellswere maintained on plastic (Chart 1C), active proliferation wasseen only when cultures were exposed to serum. Culturesexposed either to HDL alone or to transferrin and HDL did notproliferate.

Effect of Increasing Concentrations of HDL and Transferrin on the Proliferation of A-431 Carcinoma Cells Seeded inthe Total Absence of Serum on ECM-coated Dishes andMaintained under Serum-free Conditions. In order to determine the optimal concentrations of HDL and transferrin required to support the proliferation of A-431 cells maintained onECM-coated dishes and exposed to serum-free conditions,low-density (2 x 10") A-431 cells were seeded on ECM-coated

dishes and exposed to a fixed amount (10 jug/ml) of transferrinand increasing concentrations of HDL (Chart 1D) or to a fixedamount of HDL (100 Â¡igprotein per ml) and increasing concentrations of transferrin (Chart 1Â£f).As shown in Chart 1D, HDLconcentrations in the range of 25 to 100 fig protein per mlinduced cell proliferation, and saturation was observed at 250Â¡igprotein per ml. No HDL toxic effects were seen even whencultures were exposed to concentrations of HDL as high as 1mg protein per ml. Transferrin potentiated the mitogenic effectof HDL (Chart 1Â£).Minimal transferrin effects were observedat a concentration of 0.5 /ig/ml and, at 10 to 25 jug/ml, maximalalthough nonsaturating effects were observed.

Growth Rate and Life Span of Low-Density A-431 Carcinoma Cell Cultures Maintained on ECM- or Fibronectin-coated Dishes and Exposed to Medium Supplemented withTransferrin and HDL with or without the Presence of Insulin.

3 Unpublished results.

SEPTEMBER 1982 3705



D. Gospodarowicz et al.

Chart 1. A to C, comparison of the proliferation of A-431 carcinoma cells seeded intotal absence of serum on ECM (X\)- or fibro-nectin (S)-coated dishes versus plastic (C)and exposed to serum-free medium supplemented with HDL, transferrin. and insulinadded either singly or in combination versusthose of cultures exposed to 10% fetal calfserum. A-431-NBL cells (2 x 10") were

seeded onto 35-mm tissue culture dishes containing 2 ml of DME supplemented with Fun-

gizone (2.5 /ig/ml) and gentamicin (50 ng/ml).Dishes were coated with an ECM (A) or fibro-nectin (B) or were left uncoated (C). Cultureswere then exposed to transferrin (7"), insulin

(/). transferrin and insulin (TI). HDL (H), transferrin and HDL (TH), HDL and insulin (HI),transferrin. HDL, and insulin (WO, 10% fetalcalf serum (PCS), or DME alone (DÃ¬.The concentration of HDL added was 500 jig proteinper ml, that of transferrin was 10 ng per ml,and that of insulin was 2.5 ng per ml. HDL andtransferrin were added only once at Day 0;insulin was added every 4 days. After 8 daysin culture, triplicate dishes representing eachcondition were trypsinized and counted. Thestandard deviation in different determinationsdid not exceed 10% of the mean. D and Â£.effect of increasing concentrations of HDL (D)and transferrin (E) on the proliferation of A-431-NBL carcinoma cells seeded in absenceof serum on ECM-coated dishes and exposedeither to 10 jug transferrin per ml (Â£)or 100/ig HDL per ml (D). A-431-NBL cells (2 X 10"

cells/35-mm dish) were seeded as describedin 'Materials and Methods " on ECM-coated

dishes and exposed to either DME supplemented with 10 ftg transferrin per ml and increasing concentrations of HDL (D) or 100 /igprotein per ml HDL and increasing concentrations of transferrin (Â£). HDL and transferrinwere added at the indicated concentrationsonly once (Day 0). Eight days later, triplicateplates were trypsinized and counted with aCoulter Counter. The standard deviation in thedifferent determinations did not exceed 10%of the mean.

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When the growth rates of low-density A-431-NBL carcinoma

cell cultures maintained on ECM and exposed to HDL andtransferrin, to HDL-transferrin and insulin, or to 10% fetal calfserum were compared (Chart 2/0, cultures exposed toHDLitransferrin and insulin were found to have a growth ratesimilar to that of cultures exposed to fetal calf serum (averagedoubling time, 39 hr). However, after 8 days, cells stoppeddividing, and their final cell density under both conditionsdecreased during the subsequent period. Cultures exposed toHDL and transferrin had a lower growth rate than did culturesexposed to serum (average doubling time, 48 hr). However,they were still dividing actively after 10 days in culture. Thisresulted in a final cell density which was twice as high as thatof cultures exposed to optimal serum concentration (Chart 2/4).

In order to be sure that the ability of A-431 carcinoma cells

to proliferate actively when exposed to HDL and transferrinwas not restricted to a particular cell line, we have comparedthe growth kinetics of A-431-NBL cells to those of A-431carcinoma cells (A-431-FF) generously provided by Dr. Fred

Fox (University of California, Los Angeles). As shown in Chart26, when the growth kinetics of A-431-NBL cells were compared to those of A-431-FF, similar responses to HDL and

transferrin or to HDLitransferrin and insulin could be observed.

Cells proliferated equally well when exposed to HDL and transferrin, to HDLitransferrin and insulin, or to 10% fetal calf serum(average doubling time, 30 hr). As already observed with A-431-NBL cells, a drop in the final cell density of A-431-FF

cultures grown in the presence of transferriniHDL and insulin(Chart 2B) was observed after 8 days in culture. The majordifference between the 2 cell lines was in the greater prolifer-ative potential of A-431-FF. These cells could grow, although

to a limited extent, when exposed to DME alone and whenexposed to 10% serum, to transferriniHDL, or to trans-

ferrin:HDL and insulin, their final cell density was higher thanthat observed with A-431-NBL cells grown under similar conditions. The ability of A-431-FF cells to proliferate when exposed to DME supplemented with HDLitransferrin orHDLitransferrin and insulin could also be observed using dishescoated with fibronectin instead of ECM (Chart 2C). However,in the latter case, the morphological appearance of the cellswas not as healthy as that of cultures maintained on ECM-

coated dishes (Fig. 1). While cultures maintained on ECM andgrown in the presence of HDL alone (Fig. 1/4), HDL andtransferrin (Fig. 18), or fetal calf serum (Fig. 1D) were composed of large islands of healthy-looking and closely apposedcells, cultures maintained on fibronectin-coated dishes and





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Chart 2. Comparison of the proliferativerate of A-431 carcinoma cells exposed eitherto medium supplemented with 10% fetal calfserum or with transferrin:HDL with or withoutthe presence of insulin. The standard deviationin different determinations did not exceed 10%of the mean. In A, A-431 -NBL carcinoma cellswere seeded at an initial cell density of 2 X104 cells on 35-mm ECM-coated dishes in

total absence of serum as described in'Materials and Methods." Cultures were ex

posed to DME supplemented or not (â€¢)with10% fetal calf serum (10% PCS; O), transferrinand HOL (JH; d), or transferrin:HDL:insulin(THI, A). Transferrin (10 fig per ml) and HDL(500 fig protein per ml) were added only onceat the time of seeding, while insulin (2.5 fig perml) was added every 4 days. Every other day,triplicate plates were trypsinized and countedwith a Coulter Counter. In B, A-431-FF carcinoma cells (2 x 10" cells) were seeded asdescribed above on 35-mm ECM-coateddishes and exposed to DME alone or DMEsupplemented with transferrin and HDL, trans-ferrin:HDL:insulin, or 10% fetal calf serumThe concentrations of transferrin. HDL, andinsulin and the schedule of addition were asdescribed in A. InC, A-431-FF carcinoma cells(2 x IO'1 cells) were seeded as described in

A on 35-mm fibronectin-coated dishes. Cultures were exposed to DME alone or DMEsupplemented with transferrin and HDL, trans-ferrin:HDL:insulin, or 10% fetal calf serum.The concentrations of transferrin, HDL. andinsulin and the schedule of addition were asdescribed in A.

grown under similar conditions (Fig. 1, E, F, and H) werecomposed of smaller islands of cells, many of which werevacuolated. In both cases (on ECM- or fibronectin-coateddishes), cells with an aberrant (spider-like) morphology couldbe seen when cultures were grown in the presence of transfer-

rin:HDL and insulin (Fig. 1, C and G).The ability of HDL and transferrin to support the proliferation

of A-431 cells maintained on ECM-coated dishes and exposed

to a synthetic medium was not limited by the initial cell densityof the cultures, since it could be observed with cultures seededat cell densities ranging from 103 cells to 105 cells per 35-mmdish.3 Neither was it limited in time, since cells could be

passaged repeatedly in the total absence of serum (Chart 36)and still exhibited a life span similar to that of cultures exposedto optimal serum concentration (Chart 3/4). In contrast, cultures grown on fibronectin-coated dishes and exposed to

HDL:transferrin or HDLtransferrin and insulin rapidly lost theirgrowth potential and could not be passaged in the total absenceof serum for more than 8 generations (Chart 3B).

Inhibitory Effect of EGF on the Proliferation of Low-DensityA-431 Cell Cultures Maintained on ECM-coated Dishes and

Exposed to Transferrin and HDL. It has been reported byothers (15) that EGF markedly inhibits the replication of low-density A-431 cells maintained on plastic and grown in thepresence of serum. In order to see whether a similar effect ofEGF would be observed when A-431 cells were maintained onECM-coated dishes and grown in the presence of either serum-

supplemented medium or medium supplemented with HDL andtransferrin, we have analyzed its effect on cells maintainedunder such conditions (Fig. 2). EGF at concentrations rangingfrom 0.01 to 0.05 ng/ml was found to have a slight andstatistically insignificant mitogenic effect when added to cultures grown in the presence of HDL and transferrin (Fig. 28).At a concentration of 1 ng/ml, it had a growth-inhibiting activity;

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Chart 3. Comparison of the life span of A-431 carcinoma cells repeatedlypassaged at low cell density on either ECM- or fibronectin-coated dishes andexposed to either HDL and transferrin or to 10% fetal calf serum. Culture lifespan of A-431-FF carcinoma cells maintained on ECM-coated dishes (A) orfibronectin-coated dishes (A) and exposed either to DME supplemented withHDL (500 fig protein per ml) and transferrin (10 fig per ml) (HDL-trans) or to 10%fetal calf serum (PCS). HDL and transferrin were added only once, at the time ofseeding. Cells were seeded at 2 x 10" cells/35-mm ECM- (A, BÃŒor fibronectin-

coated dish (i>) either in the total absence (B, b ) or in the presence of 10% fetalcalf serum (A). Cultures were passaged every 7 to 8 days. The number ofgenerations was determined from the initial cell density 5 hr after seeding, andthe number of cells harvested was determined at each transfer. Each pointrepresents a single transfer. Roman numerals, passage number.

at concentrations above 2.5 ng/ml, it was clearly cytotoxic.Similar growth-inhibiting effects on the part of EGF were ob

served when cells were grown in the presence of serum (Fig.2A). However, the cytotoxic effect of EGF was not as markedas in the case of cultures exposed to HDL and transferrin andwas best observed at EGF concentrations above 10 ng/ml.

Requirement of Other Human Tumor Cell Lines for HDL inOrder to Proliferate Actively When Exposed to Serum-free

SEPTEMBER 1982 3707




Conditions. Previous reports have shown that human coloncarcinoma and Ewing's sarcoma cells divided actively in the

anchorage-dependent configuration when maintained on ECM-coated dishes and exposed to serum-supplemented medium

(39). In order to investigate whether the mitogenic componentspresent in serum could be HDL and transferrin, we have analyzed their effect as a function of concentration on the proliferation of low-density (2x104) colon carcinoma cells orEwing's sarcoma cell seeded on ECM-coated dishes and ex

posed to serum-free conditions. As shown in Chart 4/4, when

colon carcinoma cells were exposed to a fixed amount (25 fig/ml) of transferrin and to increasing concentrations of HDL, theirproliferation was a function of HDL concentration. At a proteinconcentration of 1000 |ug HDL per ml, optimal cell proliferationwas observed. When the effect of increasing transferrin concentrations was analyzed with cultures exposed to a fixedamount of HDL (500 /ig protein per ml), optimal cell proliferationwas observed at a transferrin concentration of 25 fig per ml(Chart 46). Likewise, in the case of Ewing's sarcoma cells

exposed to a fixed amount of transferrin (10 jug per ml), HDL ata concentration of 750 Â¡igprotein per ml induced optimal cellgrowth (Chart 4C). The concentration of transferrin required topotentiate the mitogenic effect of HDL (750 fig protein per ml)was 10 jug per ml (Chart 4D). The mitogenic effects of HDL andtransferrin on the proliferation of colon carcinoma and Ewing's

sarcoma cells were further investigated by testing their effectson the growth rate of low-density colon carcinoma and Ewing's

sarcoma cells maintained on ECM-coated dishes and exposed

Chart 4. Effect of increasing concentrations of HDL and transferrin on the proliferation of colon carcinoma or Ewing's sarcoma

cells seeded in absence of serum on ECM-coated dishes and exposed to constantamounts of either transferrin or HDL. Thestandard deviation in the different determinations did not exceed 10% of the mean. Coloncarcinoma cells (2 x 10* cells per 35-mmdish) were seeded as described in "Materials

and Methods on ECM-coated dishes and exposed to either DME supplemented with 25/ig transferrin per ml and increasing concentrations of HDL (A) or 750 fig HDL protein perml and increasing concentrations of transferrin(8). HDL and transferrin were added at theindicated concentrations only once (Day 0).Eight days later, triplicate plates were trypsin-ized and counted. Bars, final density of cultures exposed to 10% fetal calf serum (PCS).Ewing s sarcoma cells (4 x 10" cells/35-mm

dish) were seeded as described in Materialsand Methods" on ECM-coated dishes and ex

posed either to DME supplemented with 10ftg transferrin per ml and increasing concentrations of HDL (C) or 750 ng HDL protein perml and increasing concentrations of transferrin(D). HDL and transferrin were added at theindicated concentrations only once (Day 8).Eight days later, triplicate plates were trypsin-ized and counted with a Coulter counter. Bars.final density of cultures exposed to 10% fetalcalf serum.

to

to serum-free conditions. As shown in Chart 5, both coloncarcinoma cells (Chart 4,4) and Ewing's sarcoma cells (Chart

46) exposed to DME supplemented with optimal concentrations of HDL and transferrin proliferate as actively as whenthey were exposed to serum-supplemented medium and could

be serially passaged under such conditions for over 50 population doublings, the time at which the experiments were terminated.

HDL and transferrin also supported the growth of 2 mammarycarcinoma cell lines, the MCF-7 and ZR75-1 lines (Chart 6).

The final cell density of the cultures was, however, less thanthat of cells grown in the presence of serum-supplemented

medium. While insulin did not have any marked effect on thefinal cell density of MCF-7 cells grown in the presence of HDL

and transferrin (Chart 6/4), it had a marked effect on that ofZR75-1 cells (Chart 66). When the ability of HDL and transfer

rin to support the growth of rhabdomyosarcoma cells exposedto defined medium was examined, the final cell density ofcultures was similar to that of cells grown in the presence ofserum-supplemented medium (Chart 6C).

DISCUSSION

Recently, Barnes and Sato have demonstrated hormonerequirements for growth of more than 30 established cell linesby growing them in hormone-supplemented serum-free media

(for review, see Refs. 2 and 3). This approach has provided auseful method for the study of factors required for the growthof both normal and transformed cells in vitro.

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Chart 5. Comparison of the proliferate rate of colon carcinoma and Ewing's

sarcoma cells exposed either to medium supplemented with 10% fetal calf serumor with transferrin:HDL. The standard deviation in different determinations didnot exceed 10% of the mean. In A, Colon carcinoma cells were seeded at aninitial cell density of 2 x 10" cells on 35-mm ECM-coated dishes in total absenceof serum as described in "Materials and Methods." Cultures were exposed to

DME alone (A) or to DME supplemented with either 10% fetal calf serum (O) ortransferrin and HDL (O). Transferrin (25 fig per ml) and HDL (1000 fig protein perml) were added only once at the time of seeding. Every other day, triplicate plateswere trypsinized and counted with a Coulter Counter. In B, Ewing's sarcoma cells(4 X 10" cells) were seeded as described above on 35-mm ECM-coated dishes

and exposed to DME alone or to DME supplemented with transferrin and HDL or10% fetal calf serum. The concentration of transferrin was 10 fig per ml, whilethat of HDL was 750 fig protein per ml. The schedule of addition was as described

Among tumor cell lines, the growth requirements of humancolon carcinoma HC84S (32), mammary tumor MCF-7 (4) andZR-75-1 (1), and embryonal carcinoma cell line F9 (33, 34)maintained in a defined medium have been established. In allcases, insulin was the main mitogen. Among the various factorsrequired for optimal cell growth were, depending on the cellline considered, transferrin, prostaglandins, estradici, gluca-

gon, and EGF. In none of these studies or in any previousstudies was the requirement for lipoproteins examined, although they might have contributed significantly to the long-

term passage of these cell lines.The present results help to define the role of HDL in control

ling the proliferation of various human tumor cell lines, and inparticular that of the A-431 carcinoma cell line. This cell line,developed in culture by Giard ef al. (13), has an usually highnumber of EGF receptor sites (12) and has been used innumerous studies concerning the biological and biochemicaleffects of that mitogen (7-9, 23-25, 28, 31, 36). However,

little was known about the factors involved in the control of itsproliferation. The above results demonstrate that HDL is apotent mitogen for A-431 cells maintained under serum-free

conditions. The requirement for transferrin in order that anoptimal mitogenic effect of HDL be observed could reflecteither its role as an iron-carrying protein capable of deliveringthis metal to cells or its role as a detoxifying protein capable ofremoving from the medium trace amounts of toxic metals thatwould otherwise have been removed by serum proteins (3).The ways in which HDL acts as a growth-promoting agent have

not yet been defined. One possibility is that HDL, through itsability to induce /?-hydroxy-/3-methylglutaryl coenzyme A re-

ductase, would increase the synthesis of mevalonate (10, 16).This would in turn lead to increased synthesis of sterol andnonsterol products such as dolichol, ubiquinone, and isopen-

tenyl adenine, which have been shown to be of importance forcell proliferation (5).

In contrast to other human tumor cell lines (1, 4, 32), insulinwas not required by A-431 cells when cells were grown in the

presence of transferrin and HDL. This could either be due to alack of sensitivity of this cell type to insulin or reflect the factthat A-431 carcinoma cells have acquired the ability to produceinsulin-like factors. A precedent for the latter possibility can befound in the work of Todaro and De Larco (38), who havedemonstrated that in the case of viral transformation cells canacquire the ability to produce their own growth factors. Thecytotoxic effect of EGF when present at high concentrationconfirms previous reports by Gill and Lazar (15). It also demonstrates that a cytotoxic effect of EGF can be observed notonly when cells are grown on plastic and exposed to serum(15) but also when cells are grown on ECM and exposed to

too

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Chart 6. Comparison of the proliferation of mammary carcinoma cell linesMCF-7 (A), ZR-75-1 (B), and rhabdomyosarcoma cells (C) seeded in totalabsence of serum on ECM-coated dishes and exposed to medium supplementedwith HDL, transferrin, and insulin added either singly or in combination or tomedium supplemented with 10% fetal calf serum. The standard deviation indifferent determinations did not exceed 10% of the mean. In A, 2 x 10" MCF-7cells were seeded onto 35-mm ECM-coated dishes containing 2 ml of F-12:DMEmedium (1:1) supplemented with Fungizone (2.5 fig/ml) and gentamicin (50 fig/ml). Cultures were then exposed to transferrin (7); insulin (/); transferrin andinsulin (TV); HDL (Hi; transferrin and HDL (7"H); HDL and insulin (HI); transferrin.

HDL, and insulin (JHI); 10% fetal calf serum (PCS); or F-12:DME medium alone.The concentration of HDL added was 750 fig protein per ml, that of transferrinwas 25 fig per ml, and that of insulin was 0.5 jig per ml. HDL and transferrin wereadded only once at Day 0; insulin was added every 4 days. After 12 days inculture, triplicate dishes representing each condition were trypsinized andcounted. In ÃŸ4 x 104 cells were seeded onto 35-mm ECM-coated dishes

containing 2 ml of Roswell Park Memorial Institute Medium 1640 supplementedwith Fungizone (2.5 fig/ml) and gentamicin (50 fig/ml). Cultures were thenexposed to transferrin. insulin, HDL, HDL and insulin, transferrin and HDL,transferrin, HDL, and insulin. 10% fetal calf serum, or Roswell Park MemorialInstitute Medium 1640 alone (RPMI). The concentration of HDL added was 750/jg protein per ml, that of transferrin was 25 fig per ml, and that of insulin was 0.5jig per ml. HDL, transferrin, and insulin were added as described in A. After 12days in culture, triplicate dishes representing each condition were trypsinizedand counted in a hemocytometer.

In C 4 x 104 rhabdomyosarcoma cells were seeded onto 35-mm ECM-coated

dishes containing 2 ml of DME supplemented with Fungizone (2.5 fig/ml) andgentamicin (50 fig/ml). Cultures were then exposed to transferrin, HDL, transferrin and HDL. 10% fetal calf serum, or DME alone. The concentration of HDLwas 750 fig protein per ml; that of transferrin was 25 fig per ml. The schedule ofaddition was as described in A. After 8 days in culture, triplicate dishes representing each condition were trypsinized and counted with a Coulter Counter.

SEPTEMBER 1982 3709




transferrin and HDL. The mechanisms through which EGF,which has been shown to be a potent mitogen for a variety oftumor cell lines as well as for normal diploid cells (6), exerts itscytotoxic effect on A-431 cells is at present unknown.

Since A-431 cells maintained on plastic did not proliferate

actively when exposed to HDL and transferrin while they did sowhen maintained on either fibronectin- or ECM-coated dishes,

the importance of the substrate in supporting the response ofthe cell to HDL is clearly demonstrated. While no significantdifference in the ability of these substrates (either fibronectin-or ECM-coated dishes) to support cell growth in early cell

passages was apparent, significant differences began toemerge in long-term passage. This was reflected by the observation that cultures maintained on fibronectin-coated dishes

stopped growing after 8 population doublings when exposedto serum-free medium supplemented with HDL and transferrin,while cells maintained on ECM-coated dishes and exposed to

similar conditions were still growing vigorously even after 50population doublings.

The ability of HDL to support tumor cell growth under serum-free conditions is not restricted to the A-431 cell line. Coloncarcinoma, Ewing's sarcoma, and rhabdomyosarcoma cells

maintained on ECM-coated dishes could be serially passaged

and grew as well when exposed to defined medium supplemented with HDL and transferrin as when grown with serum.These 3 tumor cell lines did not require insulin in order toproliferate at an optimal rate. This observation raises the possibility that HDL and transferrin are the main serum factorswhich support the growth of these various tumor cell lines.

When the ability of HDL and transferrin to support the growthof 2 mammary carcinoma cell lines was analyzed, in both casesHDL and transferrin could support the growth of these cellsmaintained under serum-free conditions. However, the growthrate3 and final cell density reached by cultures exposed to

medium supplemented with both HDL and transferrin was lowerthan that observed with cultures exposed to serum. In the caseof ZR-75-1 mammary carcinoma cells, addition of insulin im

proved the ability of the cells to proliferate. Nevertheless, thefinal cell density of the cultures was still lower than that observed with serum. This indicates that, in the case of MCF-7and ZR-75-1 cell lines, additional factors present in serum doplay an important role in supporting cell proliferation. Thesecould be growth factors such as EGF and hormones such asglucocorticoids, thyroxine, or estrogens, which have previouslybeen shown to support the proliferation of both of these celllines maintained under serum-free conditions (1, 4).

In summary, the present study demonstrates the importanceof lipoproteins such as HDL in supporting the in vitro growth ofhuman tumor cells. The questions of whether this plasmacomponent has a similar effect in vivo and of its importancerelative to that of other factors in the neoplastic process canbe clarified only by further studies.

ACKNOWLEDGMENTS

We wish to thank Harvey Scodel for his invaluable assistance in the preparationof this manuscript.

Note Added in Proof

In a recent article Barnes (J. Cell Biol. 33. 1-4. 1982) has described thecytotoxic effect of EGF on A-431 cells maintained under serum-free conditions.

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3889, 1978.2. Barnes, D., and Sato, G. Serum-free cell culture: a unifying approach. Cell,

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SEPTEMBER 1982 3711



Fig. 1. Morphological appearance atter 8 days in culture of A-431-FF carcinoma cells maintained on either ECM- or fibronectin-coated dishes and exposed toHDL alone. HDL and transferrin. HDL:transferrin and insulin, or 10% fetal calf serum. A-431 -FF carcinoma cells (2 x 10") were seeded on either ECM-coated dishes

(/* to D) or fibronectin-coated dishes (Â£to H) as described in Chart 2, B and C. Cultures were exposed to HDL alone (500 ng protein per ml) (A and Â£);HDL andtransferrin (10 /i9 Per ml) (B and F); HDL, transferrin. and insulin (2.5 fig per ml) (C and G); or 10% fetal calf serum (D and H). After 8 days in culture, phase-contrastmicrographs (X100) were taken of each culture condition. One can observe that, in the case of cultures grown in the presence of insulin, cells with aberrantmorphology (arrows) can be seen readily.





rA

z

5V)

10%PCSÂ£

B HDL-TRANS

SEEDING

io4

4QJ

â€”10%PCS Â°

â€”-SEEDING

1-DME

J L _i_ i i10 25 "0.01 0.05 0.1

EGF(ng/ml)

0.5 2.5 5 10 25 50

Fig. 2. Effect of increasing concentrations of EGF on the proliferation of A-431 carcinoma cells maintained on ECM-coated dishes and exposed to either DMEsupplemented with 10% calf serum (Ai or to HDL:transferrin (B). A-431-NBL carcinoma cells were seeded at an initial cell density of 4 x 104 cells as described in"Materials and Methods' either in the presence of 10% fetal calf serum (PCS) (A) or in total absence of serum (B) on 35-mm ECM-coated dishes. Cultures seeded

in total absence of serum were exposed to HDL (500 /ig protein per ml) and transferrin (TRANS; 10 jig per ml). Increasing concentrations of EGF ranging from 1 to25 ng/ml in the case of cultures exposed to 10% fetal calf serum (A) and 0.01 to 50 ng/ml in the case of cultures exposed to HDLrtransferrin (B) were then addedevery other day to the cultures. Eight days later, cultures were trypsinized, and cells were counted in a Coulter Counter. Arrows, final cell density of cultures exposedto DME alone, the seeding density (SEEDING), or the final cell density of cultures exposed to either 10% fetal calf serum or HDUtransferrin (HD. C to Â£,microphotographs of cultures grown in the presence of HDL and transferrin (C) and exposed to concentrations of EGF of either 0.1 ng/ml (D) or 5 ng/nl (E). Phase-contrast, X100.

SEPTEMBER 1982 3713



1982;42:3704-3713. Cancer Res D. Gospodarowicz, G.-M. Lui and R. Gonzalez and Exposed to Defined MediumTumor Cells Maintained on Extracellular Matrix-coated Dishes High-Density Lipoproteins and the Proliferation of Human

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