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metaplastic tracheas (lanes 2, 3, and 5); however, this protein is not detected in extracts from mucociliary tracheas (Fig. 5, lanes 1 and 4). The monospecific anti-67 and -59 kDa keratin sera do not recognize any protein in extracts of these cultured trachea (data not shown). Negative results are obtained with all the preimmune sera used at the same dilution as their corresponding immune sera (data not shown).
[ 11] I n h i b i t i o n o f T u m o r C e l l G r o w t h b y R e t i n o i d s By REUBEN
LOTAN, DAFNA LOTAN, a n d PETER G . SACKS
Introduction
The growth and differentiation of various normal and malignant cells in culture are modulated (stimulated or inhibited) by retinoids) -s Most of the cultured malignant cells that have been analyzed for responsiveness to retinoids exhibit inhibition of anchorage-dependent growth and anchorage-independent growth. I-4'6-16 This chapter describes various methods used to analyze the growth inhibitory effects that retinoids exert on cultured cells.
t B. Amos and R. Lotan, this volume [23]. 2 R. Lotan, Biochim. Biophys. Acta 605, 33 (1980). 3 A. M. Jetten, in "Growth and Maturation Factors" (G. Guroff, ed.), Vol. 3, p. 251. Wiley, New York, 1985. 4 E. W. Schroder, E. Rapaport, and P. H. Black, Cancer Surv. 2, 223 (1983). 5 A. Hiragun, M. Sato, and H. Mitsui, Exp. CellRes. 145, 71 (1983). 6 L. D. Dion, J. E. Blalock, and G. E. Gifford, J. Natl. Cancerlnst. 58, 795 (1977). 7 R. Lotan and G. L. Nicolson, J. Natl. Cancer Inst. 59, 1717 (1977). s L. Part, K. Itaya, and S.-I. Hakomori, Nature (London) 273, 379 (1978). 9 D. Douer and H. P. Koeitler, J. Clin Invest. 69, 277 (1982). 10M. K. Haddox and D. H. Russell, CancerRes. 39, 2476 (1979). n L. D. Dion and G. E. Giiford, Proc. Soc. Exp. Biol. Med. 163, 510 (1980). 12R. Lotan, G. Neumann, and D. Lotan, Ann. N.Y. Acad. Sci. 359, 150 (1981). 13N. Sidell, J. Natl. Cancerlnst. 68, 589 (1981). 14F. Traganos, P. J. Higgins, C. Bueti, Z. Darzynkiewicz, and M. R. Melamed, J. Natl. Cancer Inst. 73, 205 (1984). 15R. Lotan, P. G. Sacks, D. Lotan, and W. K. HonK, Int. J. Cancer40, 224 (1987). 16R. Lotan, G. Giotta, E. Nork, and G. L. Nicolson, J. Natl. Cancer Inst. 60, 1035 (1978).
METHODS IN ENZYMOLOGY, VOL 190
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form r--vaeTved.
[ 11 ]
RETINOIDS AND TUMOR CELL GROWTH
I01
Inhibition of A n c h o r a g e - D e p e n d e n t Growth The exposure of adherent cells that grow as monolayers on plastic tissue culture dishes to medium containing retinoids often result in a decrease in growth rate (Fig. IA) and a lowered saturation density.2,3,s These effects occur in the concentration range of I n M to I0 # M of retinoids and are time-dependent and dose-dependent. DNA-synthesis is suppressed, I°-~3 and the cells are either arrested in or accumulate in the G1 phase 1°-12'14 or in the S phase of the cell cycle,3 depending on the cell type. Changes in the cell cycle occur within 12-24 hr after addition of retinoids to the culture medium, and growth inhibition can be detected 24-72 hr after treatment initiation, depending on the doubling time of the cells. Cells that form discrete colonies on plastic tissue culture dishes show a decrease in the number of colonies as well as in colony size after retinoid treatment (Fig. 1B and insets in Fig. 2A,B). 15 The removal of retinoids from the culture medium of pretreated cells often results in a reversal of the growth inhibitory effects within 24-72 hr. 2,16 The growth inhibitory effects of retinoids on anchorage-dependent growth can be determined by the following methods. Treatment of Cells with Retinoids. Retinoids are stored as a crystalline powder in an N 2 atmosphere in sealed dark containers at - 70". All procedures involving retinoids are performed under subdued fight. Before an experiment, the retinoid is dissolved in either ethanol or dimethyl sulfoxide (DMSO) at a concentration of 10 raM, and this stock solution can be stored at - 70 ° under N2 for up to 3 weeks. The retinoid is diluted from the stock solution in a series of 1 to 10 dilutions in ethanol or in DMSO to obtain solutions ranging in concentration from 0.1/zM to I0 mM. These solutions are further diluted 1 to 1000 directly in the culture medium to obtain final concentrations in the range 0.1 n M to 10 gM. A variety of growth media including modified Eagle's medium, Dulbecco's modified Eagle's medium (DMEM), Ham's F10 and F12, RPMI 1640, Waymouth's MB 752/1, and McCoy's 5A medium have been used for treatment of cells with retinoids and are usually those that best support the growth of the untreated cells. The media are often supplemented with 10% fetal calf serum, as most cells require serum for efficient growth. However, retinoids have been shown to affect cell growth in serum-free medium as well. 5 Usually, the toxicity of high doses of retinoids 17,Is is enhanced in serum-free conditions, 5,~s possibly because of the absence of serum albumin, which is known to bind retinoids. Some studies have 17 M. Audette and M. Page, Cancer Detect. Prey. 6, 497 (1983). is R. Lotan and D. Lotan, unpublished observation (1987).
B
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Fro. 1. ~-all-trans-Retinoic acid dose-dependent and time.dependent inhibition of human head and neck squamous carcinoma cell (HNSCC) proliferation, multicellular spheroid growth, and colony formation. (A) Cells were seeded in a series of 35-mm-diameter tissue culture dishes at 104 cells per dish. After 24 hr, and at 72-hr intervals thereafter, the spent medium was replaced with fresh control medium (DMEM/FI2, 1:1; 10% fetal calf serum; 0.01% dimethyl sulfoxide) or medium containing the indicated concentrations of retinoic acid. The cells were cultured at 37 ° in a humidified atmosphere composed of 94% air and 6% CO2. Dishes were removed from the incubator at each of the indicated times, the cells were detached after a brief exposure to a solution containing 0.1% trypsin and 2 m M EDTA in phosphate-buffered saline, pH 7.2, and suspended repeatedly to give a single-cell suspension, and the cells were counted using an electronic particle counter (Coulter model ZBI, Hialeah, FL). The results are presented as the means _+ S.E. of values obtained in triplicate dishes.
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RETINOIDS A N D T U M O R
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shown that the serum can alter the effect of retinoids on cells. For example, the growth ofmufine sarcoma virus-transformed 3T3 cells is not altered by retinoic acid in serum-containing medium but is stimulated by retinoic acid in serum-flee medium: However, murine S91-C2 melanoma cells are about 100 times more sensitive to rctinoic acid in serum-free medium (DMEM/F12) supplemented with insulin, transferrin, and selenious acid than in the presence of 10% fetal calf serum. Is It is advisable to allow cells to adhere and spread on the plastic dishes for 24 hr prior to the addition of retinoid-containing medium because the adhesive properties of cells can be altered by retinoids, 2 thus affecting plating efficiency. The control cultures should receive medium supplemented with no more than < 0.1% ethanol or DMSO. The cells are cultured at 37 ° in a humidified atmosphere consisting of 94% air and 6% CO2 (v/v). After 72 hr, and at 72-hr intervals thereafter, the spent medium is replaced with fresh medium with or without retinoids to replenish the retinoid, which may be depleted to 30-50% of the original concentration during a 72-hr incubation with cells. Analysis of Growth Inhibition by Cell Counting. Cells axe cultured in the absence or presence of retinoids for different times, and the cells are then detached after a brief incubation in either 2 m M EDTA or 0.1% trypsin/2 m M EDTA in calcium-free and magnesium-free phosphate-buf-
(B) Retinoie acid dose-dependent inhibition of colony formation on plastic or in agarose by 1483 HNSCC cells. To analyze the effect of retinoie acid on colony formation on plastic the cells were seeded at 3 × 103 cells per dish in a series of 100-ram-diameter dishes, and after 24 hr, and at 72-hr intervals thereal~er, the cells were treated with retinoic acid as described for (A). After 14 days the cells were fixed, stained with crystal violet, and the number of colonies containing more than 16 cells determined using an inverted microscope. To analyze the effect of retinoic acid on colony formation in semisolid agarose, the cells were suspended in 0.5% agarose at 4 × 104 cells/ml in control medium (containing 0.1% DMSO) or in medium containing retinoic acid in the concentration range between 10-9 and 10-s M, l-ml samples of the suspension were placed in 35-ram dishes on top of an agarose layer, and the dishes were placed in a 37 ° incubator. One milliliter of fresh medium, with or without retinoic acid, was placed on top of the cell-containing agarose gel after 72 hr, and at 72-hr intervals thereafter. After 14 days the number 'of colonies per dish was determined using an inverted microscope. The results are presented as the means _+ S.E. of values obtained in triplicate dishes. (C) Retinoic acid dose-dependent and time-dependent inhibition of the growth of human MDA 886Ln HNSCC multicellular spheroids. Spheroids of similar diameter were placed individually in each well of a 24-multiwell cluster plate that had been precoated with 1.25% agarose. The growth medium consisted of DMEM/F12 (1:1) containing 15% fetal calf serum and 0.01% DMSO (control) or of medium supplemented with the indicated concentrations of retinoic acid. After 72 hr, and at 72-hr intervals thereafter, the spent medium was replaced with fresh medium. At the indicated times two perpendicular diameters of the spheroids were measured using an inverted microscope with a calibrated reticle and the cell volume calculated. The values represent the means _+ S.E. of six spheroids.
i
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~
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i~
[11]
RETINOIDS A N D T U M O R CELL G R O W T H
105
fered saline, pH 7.2 (CMF-PBS). The cells are suspended in C M F - P B S by repeated pipetting to give a single-cell suspension, and the number of cells in samples of the suspensions are counted using an electronic particle counter, t6 Triplicate cultures are used for each time point and retinoid dose. The number of cells per dish is used to construct growth curves (Fig. l A) and to calculate growth inhibition according to Eq. (1) % growth inhibition -- 100 ( 1 - R/C )
(1)
where R and C represent the number of cells in treated and control cultures, respectively. Cell viability is determined by incubating cells in 0.1% (w/v) trypan blue in PBS and counting the total number of cells and the number of those excluding the stain (the viable ones), using a hemacytometer. Retinoic acid is toxic to most cells at concentrations higher than 50 #M. tT,ls If cell viability in retinoid-treated cultures is lower than 80% then it is not valid to refer to the effect of the retinoid as growth inhibition but rather as a cytotoxic effect. Cells that tend to aggregate even after trypsin treatment cannot be counted accurately by either the hemacytometer or a particle counter. In such instances it is possible to use a colodmetric cell quantitation assay. After retinoid treatment, the cell monolayers axe washed 5 times with PBS and fixed and stained by incubation for 30 rain in 0.5% crystal violet dissolved in 70% ethanol. The cell monolayers are then washed 3 times with PBS, and the stained cells are solubilized in 30% (v/v) acetic acid. The absorbance of the cell lysates at 590 nm is then measured on individual samples using a spectrophotometer or, after placing the samples in a 96-well plate, using a Titertech Multiskan reader (Flow Laboratories, McLean, VA)) s A standard curve can be obtained by staining a predetermined number of cells. The absorbances of treated cultures are then compared to those of control cultures. The colorimetric assay is also useful for microassays on small numbers of cells by plating the cells in 96-well plates and treating them with retinoids in the microwells) s Fio. 2. Morphologyof human head and neck squamous carcinomacells grown in the absence (A, C, E) or presence(B, D, F) of I/zM retinoicacid. (A, B) 1483 HNSCCcellswere grown as monolayerson plastictissueculturedishesfor 12 days,and a representativecolony from untreated(A) and treated(B) cultureswas photographed.The insetsin (A) and (B) show a 1.75-cm2 area of dishes on which the colonies were stained with crystal violet. (C, D) Coloniesof 1483 cellsformedduringa 14-daygrowthperiodin 0.5°5agarose.(E, F) Four-micron sectionsof multicellulartumor spheroids of MDA 886Ln HNSCCgrown for l0 days, then fixedin 1005formalin,processedfor histology,and stainedwithhematoxylinand eosin. Bars: 100/zm.Magnificationof insetsin (A) and (B): ×l. (A-D) Phase-contrastmicrographs; (E, F) bright-fieldmicrographs.[Figure2E,FreproducedfromP. G. Sacks,V. Oke, T. Vasty, and R. Lotan,HeadNeck ll, 219 0989).]
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Analysis of Growth Inhibition by Counting Colony Number. Growth inhibition of cells that form discrete colonies on the plastic tissue culture dishes (Fig. 2A,B) can be evaluated by comparing the number of colonies in untreated and treated cultures. The cells are seeded at clonal density (i.e., 10- 100 cells per cm 2) in plastic tissue culture dishes and incubated in control medium or in medium containing retinoids for about 2 weeks. The cells are then fixed with methanol/acetic acid (3:1, v/v), stained with 0.05% crystal violet (dissolved in 20% methanol in water), and counted under a dissecting microscope (Fig. IB)) 5 Cell Cycle Analysis. Cells are cultured in the absence or presence of retinoids in a series of tissue culture dishes, and at different times (e.g., 6, 12, 18, 24, 48, 72, 96, 120 hr) cells are detached, suspended, fixed in ethanol, and stained with a DNA staining reagent (e.g., mithramycin, propidium iodide). 1°,~2Alternative methods involve dissolution of the cell membrane by a nonionic detergent (1% Triton X-100) followed by staining the nuclei with one of the above-mentioned reagents. 14The stained cells or nuclei are subjected to analysis by a flow microfluorimeter.3,1°,12,~4 This analysis provides the distribution of fluorescence intensity per cell in the form of two peaks connected by a shoulder. The first peak represents cells in the GI phase of the cell cycle and the second peak those in the (32 plus M phases. The shoulder represents the cells that are in the S phase. Computer analysis of the data provides information on the percentage of cells in each phase of the cell cycle in untreated and retinoid-treated cultures. Accumulation in the GI phase of the cell cycle was reported for Chinese hamster ovary cells treated with retinol) ° as well as for human HeLa carcinoma cells, H murine $91-C2 melanoma cells,12 and murine Friend erythroleukcmia cells 14 treated with retinoic acid. Retinoid-treated cells may also accumulate in the S phase of the cell cycle as was found for murine Swiss 3T3 cells. 3 Analysis of DNA Synthesis. Cell growth inhibition is almost invariably associated with a decrease in DNA synthesis. To determine the effect of retinoid treatment on DNA synthesis, the cells are treated for different periods of time, and [methyl-3H]thymidine is added to the culture medium at a concentration of 1 to 5 ~Ci/ml. The cells are incubated for 1 to 18 hr, and then they are detached after trypsinization and suspended, with sampies of the suspension being analyzed for cell number or for DNA content and for the incorporation of [3H]thymidine into DNA. The latter parameter is determined by collecting a predetermined number of cells on glass fiber disks or filter paper disks and washing the filters successively with ice-cold 10% trichloroacetic acid (TCA), 5% cold TCA, and 95% (v/v) ethanol. The filters are then dried, and the radioactivity into the TCA-insoluble material is measured by scintillation spectrometry. The amount of
[11]
RETINOIDS AND TUMOR CELL GROWTH
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radioactivity incorporated per cell or per microgram DNA in untreated and treated cultures is then compared. The TCA-soluble material is also collected during the washes, and the radioactivity in this fraction is counted and used to determine whether the treatment alters thymidine uptake or thymidine incorporation into DNA, or both. Variations of this procedure have been used in several studies with retinoids.l°-~a,19
Inhibition of Anchorage-Independent Growth Several types of cells, such as lymphoid cells, grow normally in suspension. Untransformed cells derived from solid tissues require attachment to and spreading on a substrate to proliferate in culture, and they fail to proliferate if cell attachment and flattening are prevented by suspending the cells in a round configuration in semisolid media or above a nonadhesive substrate. 2°,2~ In contrast, many tumor cells are able to grow even if denied attachment because they have lost their dependence on anchorage during the malignant transformation.2°,2~ Treatment of various transformed and tumor cells with retinoids restores anchorage dependence and inhibits the ability of the cells to grow in suspension in liquid medium or in semisolid medium including methyl c e l l u l o s e , 22 agar, 23 o r a g a r o s e 12,t5~4 (Figs. 1B and 2C,2D). Some tumor cells can grow as tightly packed multicellular spheroids suspended in liquid medium. The growth of such spheroids is also suppressed by retinoids.25 Assay of Inhibition of Lymphoid Cell Growth. The inhibition by retinoids of the proliferation of cells growing in suspension is analyzed by withdrawing samples of the suspension at different times after initiation of treatment and counting the number of cells in untreated and treated cultures using an electronic particle counter as described above. Restoration of Shape-Dependent Growth Control. To obtain substrates on which cells exhibit different degrees of cell flattening, culture dishes are coated with a nonadhesive polymer, poly(2-hydroxyethyl methacrylate) [poly(HEMA), Interferon Sciences, New Brunswick, NJ]. The poly(HEMA) is suspended in 95% ethanol to make a 12% (w/v) suspension. After a vigorous mixing the suspension is centrifuged at 10,000 g ~9R. Lotan, T. Stolarsky, D. Lotan, and A. Ben-Ze'ev, Int. J. Cancer 33, 115 (1984). 2o I. MacPherson and L. Montagnier, Virology 23, 291 (1964). 2~ j. Folkman and A. Moscona, Nature (London) 273, 345 (1978). 22 L. D. Dion, J. E. Blalock, and G. E. Gifford, Exp. CellRes. 117, 15 (1978). 23 F. L. Meyskens and S. E. Salman, CancerRes. 39, 4055 (1979). R. Lotan, D. Lotan, and A. Kadouri, Exp. CellRes. 141, 79 (1982). 2s p. G. Sacks, V. Oke, T. Vasey, and R. Lotan, Head Neck 11, 219 (1989).
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(23 °, 15 min), and the supernatant is collected and used as a stock solution. This solution is diluted in absolute ethanol to obtain dilutions of 1:100, 1:200, 1:500, 1:1000, and 1:2000. To each well of a 24-well cluster plate 200 gl of each poly(HEMA) dilution is added, and the ethanol is evaporated by incubating the dishes at 37 ° for 36-38 hr. This procedure provides surfaces coated with polymer films of decreasing thickness and correspondingly increasing adhesiveness.2~ Cells grown as monolayers on plastic tissue culture dishes are pretreated with retinoids, and then they are detached and placed in unmodified wells or in poly(HEMA)-coated wells at a density of 105 cells/ml in 0.5 ml of medium with or without retinoid. After 20 hr of incubation at 37 °, the cells are pulse-labeled for 60 min with 5/zCi per well of [methyl-3H]thymidine. The cells are then suspended and analyzed for thymidine incorporation into TCA-insoluble material as described above. The radioactivity incorporated into DNA of cells incubated on unmodified plastic wells serves as control (100%). Retinoic acid treatment of various tumor cell lines suppressed their ability to synthesize DNA when cell attachment and flattening was restricted by the poly(HEMA)-coated substratum? 9 Thus, retinoic acid treatment caused the cells to behave like untransformed cells. Cells placed on poly(HEMA)-coated substratum tend to aggregate; ~9 therefore, this assay does not provide information on the response of individual clones within the population. Inhibition of Colony Formation in Semisolid Medium. There are several methods for suspending cells and maintaining them at clonal density using semisolid media? 2,22-24 One such medium is methy cellulose. Cells are suspended at 103 to 105 cells/ml, depending on their colony-forming efficiency, in medium containing 1.17% (w/v) methyl cellulose, 10% serum, and 0.1% ethanol or 0.1% DMSO or different concentrations of a retinoid. Samples containing 2 ml cell suspension are placed in each well of a multiwell plate or into 35-mm-diameter tissue culture dishes, in which 2 ml of 0.5% agar (w/v) has been previously placed, and allowed to gel. The plates or dishes are then incubated at 37 ° in a humidified atmosphere consisting of 94% air and 6% CO2 for 14 days, and the number of colonies is determined using an inverted microscope.22 Agar has also been used as a semisolid medium to analyze the effect of retinoids on tumor cells and primary cells derived from fresh tumor biopsies. The cells are suspended at 5 × 105 cells/ml in 0.3% agar in medium containing serum and either solvent or retinoid (at twice the desired final concentration), and 1-ml samples of the suspension are placed in 35-mm plastic dishes on top of a precast underlayer of I ml of 0.5% agar in serum-containing medium. The dishes are then incubated for 14 days, and the number of colonies in treated cultures is compared to the number in control cultures. 9,23 In this
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procedure the retinoid is added only at the beginning of the culture and is not replenished, although it is known that the retinoid is degraded within a few days. Another method utilizes low-temperature-gelling agarose, which provires improved colon-forming efficiency for certain cell types. Cells are suspended at 103 to l0 s cells/ml, depending on their colony-forming efficiency, in 0.5% agarose (w/v) in medium containing serum and solvent or retinoid, and 1-ml samples are placed in 35-ram-diameter plastic dishes on top of a layer of 1% agarose (in the same medium), which has been allowed to gel previously. The dishes are kept at 4 ° for 10 rain to gel the cell-containing agarose, and then the dishes are incubated at 37 ° as described above. During this incubation the cultures are reexposed to fresh medium with or without retinoids every 72 hr by initially placing and subsequently replacing 1 ml of medium on top of the cell-containing agarose layer. After 14 days, the number colonies with over 50 cells per colony is determined under an inverted microscope. 12,24 The observation of colonies can be facilitated by staining the viable cells. To stain the colonies in agarose the medium is removed, and 1 ml of a solution containing 1 mg of the tetrazolium salt 2-(p-iodophenyl)-3-(nitrophenyl)-5-phenyltetrazoliumchloride per milliliter of 0.9% sodium chloride solution is placed over the agarose. After an additional 24-hr incubation, the colonies stained bright red with the precipitated formazan are counted. An example for the retinoic acid dose-dependent suppression of colony formation by human squamous carcinoma cells is presented in Fig. 1B, and the morphology of such colonies (unstained) is shown in Fig. 2C,D. Suppression of Growth of Multicellular Spheroids. There are some tumor cells that can grow in suspension as tightly-packed three-dimensional multicellular spheroids. The volume of such spheroids increases over time, and they are used as models for preclinical study of chemotherapeutic agents, including retinoids.:s Spheroids are formed by suspending cells from monolayer cultures at the logarithmic growth phase, placing them on plastic petri dishes which had been precoated with 1.25% agarose in normal serum-containing medium, and incubating them for 3 days at 37 °. Small spheroidal aggregates formed during this time are transferred to spinner flasks, and the flasks are gassed with 5% CO 2 and incubated at 37 °. For growth inhibition experiments, spheroids are sized using a calibrated reticle under a dissecting microscope, and spheroids of a similar initial diameter are picked and placed in individual wells of a 24-well plate, which had been coated with agarose as above, in control medium or in medium containing different concentrations of retinoids. On the first day and at 72-hr intervals, when the medium is changed, spheroids are sized by measuring two perpendicular diameters (a and b) of the spheroids using the
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microscope and reticle. The volume of the spheroids is calculated according to Eq. (2) (2)
V---- ~7~(fa-b/2) 3
as described elsewhere.26 Using this procedure it is possible to obtain information on the time and dose dependence of growth inhibition of the spheroids by retinoids as demonstrated in Fig. 1C. The spheroids can also be fixed and processed for histological analysis as shown in Fig. 2E,F. Concluding Remarks Retinoids inhibit the growth of numerous tumor cells and suppress the expression of the transformed phenotype as represented by anchorage-independent growth. The effective concentrations of retinoids for inhibition of tumor cell growth in culture are often pharmacological and not physiological. However, quite a large number of tumor cells are inhibited considerably even at physiological dose (l # M for retinol and l0 nM for retinoic acid). Often cells that are only marginally inhibited in monolayer culture show marked inhibition of anchorage-independent growth in agarose, suggesting that the inhibition of anchorage-independent growth is a more sensitive assay for the suppression of the growth of tumor cells by retinoids. 24T. Nederman and P. Twentyman, Rec. Results CancerRes. 95, 84 (1984).
[12] M a i n t e n a n c e
and Use of F9 Teratocarcinoma
By ANDREW L. DARROW, RICHARD J.
Cells
RICKLES, and SIDNEY STRICKLAND
Introduction F9 cells are an established cell line of mouse embryonal carcinoma eels that differentiate spontaneously at very low frequency. Although F9 cells are routinely maintained and passaged as stem cells, treatment with retinoic acid (RA) induces differentiation in culture, producing cells that resemble primitive extraembryonic endoderm of the mouse embryo. RA-treated F9 eels are bipotential: aggregation results in the appearance of visceral endoderm,2 whereas further treatment with cyclic AMP (RA/ S. Strickland and V. Mahdavi, Cell (Cambridge, Mass.) 15, 393 (1978). 2 B. L. M. Hogan, A. Taylor, and E. Adamson, Nature(London) 291, 235 (1981).
METHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Pre~ Inc. All rights of reproductionin any formre~-rved.
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metaplastic tracheas (lanes 2, 3, and 5); however, this protein is not detected in extracts from mucociliary tracheas (Fig. 5, lanes 1 and 4). The monospecific anti-67 and -59 kDa keratin sera do not recognize any protein in extracts of these cultured trachea (data not shown). Negative results are obtained with all the preimmune sera used at the same dilution as their corresponding immune sera (data not shown).
[ 11] I n h i b i t i o n o f T u m o r C e l l G r o w t h b y R e t i n o i d s By REUBEN
LOTAN, DAFNA LOTAN, a n d PETER G . SACKS
Introduction
The growth and differentiation of various normal and malignant cells in culture are modulated (stimulated or inhibited) by retinoids) -s Most of the cultured malignant cells that have been analyzed for responsiveness to retinoids exhibit inhibition of anchorage-dependent growth and anchorage-independent growth. I-4'6-16 This chapter describes various methods used to analyze the growth inhibitory effects that retinoids exert on cultured cells.
t B. Amos and R. Lotan, this volume [23]. 2 R. Lotan, Biochim. Biophys. Acta 605, 33 (1980). 3 A. M. Jetten, in "Growth and Maturation Factors" (G. Guroff, ed.), Vol. 3, p. 251. Wiley, New York, 1985. 4 E. W. Schroder, E. Rapaport, and P. H. Black, Cancer Surv. 2, 223 (1983). 5 A. Hiragun, M. Sato, and H. Mitsui, Exp. CellRes. 145, 71 (1983). 6 L. D. Dion, J. E. Blalock, and G. E. Gifford, J. Natl. Cancerlnst. 58, 795 (1977). 7 R. Lotan and G. L. Nicolson, J. Natl. Cancer Inst. 59, 1717 (1977). s L. Part, K. Itaya, and S.-I. Hakomori, Nature (London) 273, 379 (1978). 9 D. Douer and H. P. Koeitler, J. Clin Invest. 69, 277 (1982). 10M. K. Haddox and D. H. Russell, CancerRes. 39, 2476 (1979). n L. D. Dion and G. E. Giiford, Proc. Soc. Exp. Biol. Med. 163, 510 (1980). 12R. Lotan, G. Neumann, and D. Lotan, Ann. N.Y. Acad. Sci. 359, 150 (1981). 13N. Sidell, J. Natl. Cancerlnst. 68, 589 (1981). 14F. Traganos, P. J. Higgins, C. Bueti, Z. Darzynkiewicz, and M. R. Melamed, J. Natl. Cancer Inst. 73, 205 (1984). 15R. Lotan, P. G. Sacks, D. Lotan, and W. K. HonK, Int. J. Cancer40, 224 (1987). 16R. Lotan, G. Giotta, E. Nork, and G. L. Nicolson, J. Natl. Cancer Inst. 60, 1035 (1978).
METHODS IN ENZYMOLOGY, VOL 190
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form r--vaeTved.
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RETINOIDS AND TUMOR CELL GROWTH
I01
Inhibition of A n c h o r a g e - D e p e n d e n t Growth The exposure of adherent cells that grow as monolayers on plastic tissue culture dishes to medium containing retinoids often result in a decrease in growth rate (Fig. IA) and a lowered saturation density.2,3,s These effects occur in the concentration range of I n M to I0 # M of retinoids and are time-dependent and dose-dependent. DNA-synthesis is suppressed, I°-~3 and the cells are either arrested in or accumulate in the G1 phase 1°-12'14 or in the S phase of the cell cycle,3 depending on the cell type. Changes in the cell cycle occur within 12-24 hr after addition of retinoids to the culture medium, and growth inhibition can be detected 24-72 hr after treatment initiation, depending on the doubling time of the cells. Cells that form discrete colonies on plastic tissue culture dishes show a decrease in the number of colonies as well as in colony size after retinoid treatment (Fig. 1B and insets in Fig. 2A,B). 15 The removal of retinoids from the culture medium of pretreated cells often results in a reversal of the growth inhibitory effects within 24-72 hr. 2,16 The growth inhibitory effects of retinoids on anchorage-dependent growth can be determined by the following methods. Treatment of Cells with Retinoids. Retinoids are stored as a crystalline powder in an N 2 atmosphere in sealed dark containers at - 70". All procedures involving retinoids are performed under subdued fight. Before an experiment, the retinoid is dissolved in either ethanol or dimethyl sulfoxide (DMSO) at a concentration of 10 raM, and this stock solution can be stored at - 70 ° under N2 for up to 3 weeks. The retinoid is diluted from the stock solution in a series of 1 to 10 dilutions in ethanol or in DMSO to obtain solutions ranging in concentration from 0.1/zM to I0 mM. These solutions are further diluted 1 to 1000 directly in the culture medium to obtain final concentrations in the range 0.1 n M to 10 gM. A variety of growth media including modified Eagle's medium, Dulbecco's modified Eagle's medium (DMEM), Ham's F10 and F12, RPMI 1640, Waymouth's MB 752/1, and McCoy's 5A medium have been used for treatment of cells with retinoids and are usually those that best support the growth of the untreated cells. The media are often supplemented with 10% fetal calf serum, as most cells require serum for efficient growth. However, retinoids have been shown to affect cell growth in serum-free medium as well. 5 Usually, the toxicity of high doses of retinoids 17,Is is enhanced in serum-free conditions, 5,~s possibly because of the absence of serum albumin, which is known to bind retinoids. Some studies have 17 M. Audette and M. Page, Cancer Detect. Prey. 6, 497 (1983). is R. Lotan and D. Lotan, unpublished observation (1987).
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Fro. 1. ~-all-trans-Retinoic acid dose-dependent and time.dependent inhibition of human head and neck squamous carcinoma cell (HNSCC) proliferation, multicellular spheroid growth, and colony formation. (A) Cells were seeded in a series of 35-mm-diameter tissue culture dishes at 104 cells per dish. After 24 hr, and at 72-hr intervals thereafter, the spent medium was replaced with fresh control medium (DMEM/FI2, 1:1; 10% fetal calf serum; 0.01% dimethyl sulfoxide) or medium containing the indicated concentrations of retinoic acid. The cells were cultured at 37 ° in a humidified atmosphere composed of 94% air and 6% CO2. Dishes were removed from the incubator at each of the indicated times, the cells were detached after a brief exposure to a solution containing 0.1% trypsin and 2 m M EDTA in phosphate-buffered saline, pH 7.2, and suspended repeatedly to give a single-cell suspension, and the cells were counted using an electronic particle counter (Coulter model ZBI, Hialeah, FL). The results are presented as the means _+ S.E. of values obtained in triplicate dishes.
[11]
RETINOIDS A N D T U M O R
CELL G R O W T H
103
shown that the serum can alter the effect of retinoids on cells. For example, the growth ofmufine sarcoma virus-transformed 3T3 cells is not altered by retinoic acid in serum-containing medium but is stimulated by retinoic acid in serum-flee medium: However, murine S91-C2 melanoma cells are about 100 times more sensitive to rctinoic acid in serum-free medium (DMEM/F12) supplemented with insulin, transferrin, and selenious acid than in the presence of 10% fetal calf serum. Is It is advisable to allow cells to adhere and spread on the plastic dishes for 24 hr prior to the addition of retinoid-containing medium because the adhesive properties of cells can be altered by retinoids, 2 thus affecting plating efficiency. The control cultures should receive medium supplemented with no more than < 0.1% ethanol or DMSO. The cells are cultured at 37 ° in a humidified atmosphere consisting of 94% air and 6% CO2 (v/v). After 72 hr, and at 72-hr intervals thereafter, the spent medium is replaced with fresh medium with or without retinoids to replenish the retinoid, which may be depleted to 30-50% of the original concentration during a 72-hr incubation with cells. Analysis of Growth Inhibition by Cell Counting. Cells axe cultured in the absence or presence of retinoids for different times, and the cells are then detached after a brief incubation in either 2 m M EDTA or 0.1% trypsin/2 m M EDTA in calcium-free and magnesium-free phosphate-buf-
(B) Retinoie acid dose-dependent inhibition of colony formation on plastic or in agarose by 1483 HNSCC cells. To analyze the effect of retinoie acid on colony formation on plastic the cells were seeded at 3 × 103 cells per dish in a series of 100-ram-diameter dishes, and after 24 hr, and at 72-hr intervals thereal~er, the cells were treated with retinoic acid as described for (A). After 14 days the cells were fixed, stained with crystal violet, and the number of colonies containing more than 16 cells determined using an inverted microscope. To analyze the effect of retinoic acid on colony formation in semisolid agarose, the cells were suspended in 0.5% agarose at 4 × 104 cells/ml in control medium (containing 0.1% DMSO) or in medium containing retinoic acid in the concentration range between 10-9 and 10-s M, l-ml samples of the suspension were placed in 35-ram dishes on top of an agarose layer, and the dishes were placed in a 37 ° incubator. One milliliter of fresh medium, with or without retinoic acid, was placed on top of the cell-containing agarose gel after 72 hr, and at 72-hr intervals thereafter. After 14 days the number 'of colonies per dish was determined using an inverted microscope. The results are presented as the means _+ S.E. of values obtained in triplicate dishes. (C) Retinoic acid dose-dependent and time-dependent inhibition of the growth of human MDA 886Ln HNSCC multicellular spheroids. Spheroids of similar diameter were placed individually in each well of a 24-multiwell cluster plate that had been precoated with 1.25% agarose. The growth medium consisted of DMEM/F12 (1:1) containing 15% fetal calf serum and 0.01% DMSO (control) or of medium supplemented with the indicated concentrations of retinoic acid. After 72 hr, and at 72-hr intervals thereafter, the spent medium was replaced with fresh medium. At the indicated times two perpendicular diameters of the spheroids were measured using an inverted microscope with a calibrated reticle and the cell volume calculated. The values represent the means _+ S.E. of six spheroids.
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[11]
RETINOIDS A N D T U M O R CELL G R O W T H
105
fered saline, pH 7.2 (CMF-PBS). The cells are suspended in C M F - P B S by repeated pipetting to give a single-cell suspension, and the number of cells in samples of the suspensions are counted using an electronic particle counter, t6 Triplicate cultures are used for each time point and retinoid dose. The number of cells per dish is used to construct growth curves (Fig. l A) and to calculate growth inhibition according to Eq. (1) % growth inhibition -- 100 ( 1 - R/C )
(1)
where R and C represent the number of cells in treated and control cultures, respectively. Cell viability is determined by incubating cells in 0.1% (w/v) trypan blue in PBS and counting the total number of cells and the number of those excluding the stain (the viable ones), using a hemacytometer. Retinoic acid is toxic to most cells at concentrations higher than 50 #M. tT,ls If cell viability in retinoid-treated cultures is lower than 80% then it is not valid to refer to the effect of the retinoid as growth inhibition but rather as a cytotoxic effect. Cells that tend to aggregate even after trypsin treatment cannot be counted accurately by either the hemacytometer or a particle counter. In such instances it is possible to use a colodmetric cell quantitation assay. After retinoid treatment, the cell monolayers axe washed 5 times with PBS and fixed and stained by incubation for 30 rain in 0.5% crystal violet dissolved in 70% ethanol. The cell monolayers are then washed 3 times with PBS, and the stained cells are solubilized in 30% (v/v) acetic acid. The absorbance of the cell lysates at 590 nm is then measured on individual samples using a spectrophotometer or, after placing the samples in a 96-well plate, using a Titertech Multiskan reader (Flow Laboratories, McLean, VA)) s A standard curve can be obtained by staining a predetermined number of cells. The absorbances of treated cultures are then compared to those of control cultures. The colorimetric assay is also useful for microassays on small numbers of cells by plating the cells in 96-well plates and treating them with retinoids in the microwells) s Fio. 2. Morphologyof human head and neck squamous carcinomacells grown in the absence (A, C, E) or presence(B, D, F) of I/zM retinoicacid. (A, B) 1483 HNSCCcellswere grown as monolayerson plastictissueculturedishesfor 12 days,and a representativecolony from untreated(A) and treated(B) cultureswas photographed.The insetsin (A) and (B) show a 1.75-cm2 area of dishes on which the colonies were stained with crystal violet. (C, D) Coloniesof 1483 cellsformedduringa 14-daygrowthperiodin 0.5°5agarose.(E, F) Four-micron sectionsof multicellulartumor spheroids of MDA 886Ln HNSCCgrown for l0 days, then fixedin 1005formalin,processedfor histology,and stainedwithhematoxylinand eosin. Bars: 100/zm.Magnificationof insetsin (A) and (B): ×l. (A-D) Phase-contrastmicrographs; (E, F) bright-fieldmicrographs.[Figure2E,FreproducedfromP. G. Sacks,V. Oke, T. Vasty, and R. Lotan,HeadNeck ll, 219 0989).]
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Analysis of Growth Inhibition by Counting Colony Number. Growth inhibition of cells that form discrete colonies on the plastic tissue culture dishes (Fig. 2A,B) can be evaluated by comparing the number of colonies in untreated and treated cultures. The cells are seeded at clonal density (i.e., 10- 100 cells per cm 2) in plastic tissue culture dishes and incubated in control medium or in medium containing retinoids for about 2 weeks. The cells are then fixed with methanol/acetic acid (3:1, v/v), stained with 0.05% crystal violet (dissolved in 20% methanol in water), and counted under a dissecting microscope (Fig. IB)) 5 Cell Cycle Analysis. Cells are cultured in the absence or presence of retinoids in a series of tissue culture dishes, and at different times (e.g., 6, 12, 18, 24, 48, 72, 96, 120 hr) cells are detached, suspended, fixed in ethanol, and stained with a DNA staining reagent (e.g., mithramycin, propidium iodide). 1°,~2Alternative methods involve dissolution of the cell membrane by a nonionic detergent (1% Triton X-100) followed by staining the nuclei with one of the above-mentioned reagents. 14The stained cells or nuclei are subjected to analysis by a flow microfluorimeter.3,1°,12,~4 This analysis provides the distribution of fluorescence intensity per cell in the form of two peaks connected by a shoulder. The first peak represents cells in the GI phase of the cell cycle and the second peak those in the (32 plus M phases. The shoulder represents the cells that are in the S phase. Computer analysis of the data provides information on the percentage of cells in each phase of the cell cycle in untreated and retinoid-treated cultures. Accumulation in the GI phase of the cell cycle was reported for Chinese hamster ovary cells treated with retinol) ° as well as for human HeLa carcinoma cells, H murine $91-C2 melanoma cells,12 and murine Friend erythroleukcmia cells 14 treated with retinoic acid. Retinoid-treated cells may also accumulate in the S phase of the cell cycle as was found for murine Swiss 3T3 cells. 3 Analysis of DNA Synthesis. Cell growth inhibition is almost invariably associated with a decrease in DNA synthesis. To determine the effect of retinoid treatment on DNA synthesis, the cells are treated for different periods of time, and [methyl-3H]thymidine is added to the culture medium at a concentration of 1 to 5 ~Ci/ml. The cells are incubated for 1 to 18 hr, and then they are detached after trypsinization and suspended, with sampies of the suspension being analyzed for cell number or for DNA content and for the incorporation of [3H]thymidine into DNA. The latter parameter is determined by collecting a predetermined number of cells on glass fiber disks or filter paper disks and washing the filters successively with ice-cold 10% trichloroacetic acid (TCA), 5% cold TCA, and 95% (v/v) ethanol. The filters are then dried, and the radioactivity into the TCA-insoluble material is measured by scintillation spectrometry. The amount of
[11]
RETINOIDS AND TUMOR CELL GROWTH
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radioactivity incorporated per cell or per microgram DNA in untreated and treated cultures is then compared. The TCA-soluble material is also collected during the washes, and the radioactivity in this fraction is counted and used to determine whether the treatment alters thymidine uptake or thymidine incorporation into DNA, or both. Variations of this procedure have been used in several studies with retinoids.l°-~a,19
Inhibition of Anchorage-Independent Growth Several types of cells, such as lymphoid cells, grow normally in suspension. Untransformed cells derived from solid tissues require attachment to and spreading on a substrate to proliferate in culture, and they fail to proliferate if cell attachment and flattening are prevented by suspending the cells in a round configuration in semisolid media or above a nonadhesive substrate. 2°,2~ In contrast, many tumor cells are able to grow even if denied attachment because they have lost their dependence on anchorage during the malignant transformation.2°,2~ Treatment of various transformed and tumor cells with retinoids restores anchorage dependence and inhibits the ability of the cells to grow in suspension in liquid medium or in semisolid medium including methyl c e l l u l o s e , 22 agar, 23 o r a g a r o s e 12,t5~4 (Figs. 1B and 2C,2D). Some tumor cells can grow as tightly packed multicellular spheroids suspended in liquid medium. The growth of such spheroids is also suppressed by retinoids.25 Assay of Inhibition of Lymphoid Cell Growth. The inhibition by retinoids of the proliferation of cells growing in suspension is analyzed by withdrawing samples of the suspension at different times after initiation of treatment and counting the number of cells in untreated and treated cultures using an electronic particle counter as described above. Restoration of Shape-Dependent Growth Control. To obtain substrates on which cells exhibit different degrees of cell flattening, culture dishes are coated with a nonadhesive polymer, poly(2-hydroxyethyl methacrylate) [poly(HEMA), Interferon Sciences, New Brunswick, NJ]. The poly(HEMA) is suspended in 95% ethanol to make a 12% (w/v) suspension. After a vigorous mixing the suspension is centrifuged at 10,000 g ~9R. Lotan, T. Stolarsky, D. Lotan, and A. Ben-Ze'ev, Int. J. Cancer 33, 115 (1984). 2o I. MacPherson and L. Montagnier, Virology 23, 291 (1964). 2~ j. Folkman and A. Moscona, Nature (London) 273, 345 (1978). 22 L. D. Dion, J. E. Blalock, and G. E. Gifford, Exp. CellRes. 117, 15 (1978). 23 F. L. Meyskens and S. E. Salman, CancerRes. 39, 4055 (1979). R. Lotan, D. Lotan, and A. Kadouri, Exp. CellRes. 141, 79 (1982). 2s p. G. Sacks, V. Oke, T. Vasey, and R. Lotan, Head Neck 11, 219 (1989).
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(23 °, 15 min), and the supernatant is collected and used as a stock solution. This solution is diluted in absolute ethanol to obtain dilutions of 1:100, 1:200, 1:500, 1:1000, and 1:2000. To each well of a 24-well cluster plate 200 gl of each poly(HEMA) dilution is added, and the ethanol is evaporated by incubating the dishes at 37 ° for 36-38 hr. This procedure provides surfaces coated with polymer films of decreasing thickness and correspondingly increasing adhesiveness.2~ Cells grown as monolayers on plastic tissue culture dishes are pretreated with retinoids, and then they are detached and placed in unmodified wells or in poly(HEMA)-coated wells at a density of 105 cells/ml in 0.5 ml of medium with or without retinoid. After 20 hr of incubation at 37 °, the cells are pulse-labeled for 60 min with 5/zCi per well of [methyl-3H]thymidine. The cells are then suspended and analyzed for thymidine incorporation into TCA-insoluble material as described above. The radioactivity incorporated into DNA of cells incubated on unmodified plastic wells serves as control (100%). Retinoic acid treatment of various tumor cell lines suppressed their ability to synthesize DNA when cell attachment and flattening was restricted by the poly(HEMA)-coated substratum? 9 Thus, retinoic acid treatment caused the cells to behave like untransformed cells. Cells placed on poly(HEMA)-coated substratum tend to aggregate; ~9 therefore, this assay does not provide information on the response of individual clones within the population. Inhibition of Colony Formation in Semisolid Medium. There are several methods for suspending cells and maintaining them at clonal density using semisolid media? 2,22-24 One such medium is methy cellulose. Cells are suspended at 103 to 105 cells/ml, depending on their colony-forming efficiency, in medium containing 1.17% (w/v) methyl cellulose, 10% serum, and 0.1% ethanol or 0.1% DMSO or different concentrations of a retinoid. Samples containing 2 ml cell suspension are placed in each well of a multiwell plate or into 35-mm-diameter tissue culture dishes, in which 2 ml of 0.5% agar (w/v) has been previously placed, and allowed to gel. The plates or dishes are then incubated at 37 ° in a humidified atmosphere consisting of 94% air and 6% CO2 for 14 days, and the number of colonies is determined using an inverted microscope.22 Agar has also been used as a semisolid medium to analyze the effect of retinoids on tumor cells and primary cells derived from fresh tumor biopsies. The cells are suspended at 5 × 105 cells/ml in 0.3% agar in medium containing serum and either solvent or retinoid (at twice the desired final concentration), and 1-ml samples of the suspension are placed in 35-mm plastic dishes on top of a precast underlayer of I ml of 0.5% agar in serum-containing medium. The dishes are then incubated for 14 days, and the number of colonies in treated cultures is compared to the number in control cultures. 9,23 In this
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RETINOIDS AND T U M O R CELL GROWTH
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procedure the retinoid is added only at the beginning of the culture and is not replenished, although it is known that the retinoid is degraded within a few days. Another method utilizes low-temperature-gelling agarose, which provires improved colon-forming efficiency for certain cell types. Cells are suspended at 103 to l0 s cells/ml, depending on their colony-forming efficiency, in 0.5% agarose (w/v) in medium containing serum and solvent or retinoid, and 1-ml samples are placed in 35-ram-diameter plastic dishes on top of a layer of 1% agarose (in the same medium), which has been allowed to gel previously. The dishes are kept at 4 ° for 10 rain to gel the cell-containing agarose, and then the dishes are incubated at 37 ° as described above. During this incubation the cultures are reexposed to fresh medium with or without retinoids every 72 hr by initially placing and subsequently replacing 1 ml of medium on top of the cell-containing agarose layer. After 14 days, the number colonies with over 50 cells per colony is determined under an inverted microscope. 12,24 The observation of colonies can be facilitated by staining the viable cells. To stain the colonies in agarose the medium is removed, and 1 ml of a solution containing 1 mg of the tetrazolium salt 2-(p-iodophenyl)-3-(nitrophenyl)-5-phenyltetrazoliumchloride per milliliter of 0.9% sodium chloride solution is placed over the agarose. After an additional 24-hr incubation, the colonies stained bright red with the precipitated formazan are counted. An example for the retinoic acid dose-dependent suppression of colony formation by human squamous carcinoma cells is presented in Fig. 1B, and the morphology of such colonies (unstained) is shown in Fig. 2C,D. Suppression of Growth of Multicellular Spheroids. There are some tumor cells that can grow in suspension as tightly-packed three-dimensional multicellular spheroids. The volume of such spheroids increases over time, and they are used as models for preclinical study of chemotherapeutic agents, including retinoids.:s Spheroids are formed by suspending cells from monolayer cultures at the logarithmic growth phase, placing them on plastic petri dishes which had been precoated with 1.25% agarose in normal serum-containing medium, and incubating them for 3 days at 37 °. Small spheroidal aggregates formed during this time are transferred to spinner flasks, and the flasks are gassed with 5% CO 2 and incubated at 37 °. For growth inhibition experiments, spheroids are sized using a calibrated reticle under a dissecting microscope, and spheroids of a similar initial diameter are picked and placed in individual wells of a 24-well plate, which had been coated with agarose as above, in control medium or in medium containing different concentrations of retinoids. On the first day and at 72-hr intervals, when the medium is changed, spheroids are sized by measuring two perpendicular diameters (a and b) of the spheroids using the
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microscope and reticle. The volume of the spheroids is calculated according to Eq. (2) (2)
V---- ~7~(fa-b/2) 3
as described elsewhere.26 Using this procedure it is possible to obtain information on the time and dose dependence of growth inhibition of the spheroids by retinoids as demonstrated in Fig. 1C. The spheroids can also be fixed and processed for histological analysis as shown in Fig. 2E,F. Concluding Remarks Retinoids inhibit the growth of numerous tumor cells and suppress the expression of the transformed phenotype as represented by anchorage-independent growth. The effective concentrations of retinoids for inhibition of tumor cell growth in culture are often pharmacological and not physiological. However, quite a large number of tumor cells are inhibited considerably even at physiological dose (l # M for retinol and l0 nM for retinoic acid). Often cells that are only marginally inhibited in monolayer culture show marked inhibition of anchorage-independent growth in agarose, suggesting that the inhibition of anchorage-independent growth is a more sensitive assay for the suppression of the growth of tumor cells by retinoids. 24T. Nederman and P. Twentyman, Rec. Results CancerRes. 95, 84 (1984).
[12] M a i n t e n a n c e
and Use of F9 Teratocarcinoma
By ANDREW L. DARROW, RICHARD J.
Cells
RICKLES, and SIDNEY STRICKLAND
Introduction F9 cells are an established cell line of mouse embryonal carcinoma eels that differentiate spontaneously at very low frequency. Although F9 cells are routinely maintained and passaged as stem cells, treatment with retinoic acid (RA) induces differentiation in culture, producing cells that resemble primitive extraembryonic endoderm of the mouse embryo. RA-treated F9 eels are bipotential: aggregation results in the appearance of visceral endoderm,2 whereas further treatment with cyclic AMP (RA/ S. Strickland and V. Mahdavi, Cell (Cambridge, Mass.) 15, 393 (1978). 2 B. L. M. Hogan, A. Taylor, and E. Adamson, Nature(London) 291, 235 (1981).
METHODS IN ENZYMOLOGY, VOL. 190
Copyright © 1990 by Academic Pre~ Inc. All rights of reproductionin any formre~-rved.
