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[35] D i f f e r e n t i a t i o n o f E m b r y o n a l C a r c i n o m a Cells in R e s p o n s e to R e t i n o ! d s
By PATRICIO ABAaZ6A and MICHAEL I. SHERMAN Introduction Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, offer an attractive experimental model for studying differentiation because they resemble early embryonic stem cells.l Embryonal carcinoma cells were initially isolated from primary cultures of cells derived from teratocarcinomas, and from these many clonal cell lines have been developed. The propensity for differentiation of cells from these lines can be assessed in vivo; such studies have indicated a broad spectrum of differentiation potential among the lines. 2 When so-called pluripotent EC cells (e.g., PCC3, PC-13) are injected subcutaneously into syngeneic mice, they give rise to teratocarcinomas which typically contain many differentiated cell types that can represent all three primary germ layers. At the other extreme, so-called nullipotent EC cells (e.g., Nulli-SCC1, F9) show little or no histological evidence of differentiation. In fact, this classification is artificial insofar as there is probably a continuum of potential for differentiation in tumors if one considers large numbers of EC lines. In tissue culture, the ability of EC cells to differentiate also varies markedly. Cells from most EC lines remain undifferentiated or differentiate at low frequency when growing exponentially. Some lines differentiate readily on reaching confluence. When EC cells are prevented from attaching to the substratum, they form multicellular aggregates, and under these conditions cells from some of the lines undergo differentiation. 3 A variety of chemicals have been shown to induce EC cell differentiation; retinoids are among the most potent chemical inducers of EC cell differentiation, with effective concentrations typically in the nanomolar range. Even EC cells such as NulIi-SCC1 and F9, which differentiate poorly in tumors, do so readily in culture in response to retinoids. 4,5 Because the G. R. Martin, Science 2119, 768 (1980). 2 G. R. Martin, in "Teratocarcinoma Stem Cells" (L. M. Silver, G. R. Martin, and S. Strickland, eds.), p. 690. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 3 M. I. Sherman, in "Teratomas and Differentiation" (M. I. Sherman and D. Solter, eds.), p. 189. Academic Press, New York, 1975. 4 S. Strickland and V. Mahdavi, Cell (Cambridge, Mass.) 15, 393 (1978). 5 A. M. Jetten, M. E. R. Jetten, and M. I. Sherman, Exp. Cell Res. 124, 381 (1979).
METHODS IN ENZYMOLOGY, VOL. 189
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mechanisms by which retinoids induce differentiation of EC cells might be relevant to their biochemical and biological effects on a large variety of cell types, and because differentiation is easily monitored in EC cells, this cell system is useful for studying retinoid action. We describe below techniques for the culture of EC cells from established lines and for evaluating the differentiation of these cells in response to retinoids. Culture of Embryonal Carcinoma Cells Although human EC cells have been cultured, 2 as a rule they are less responsive to retinoids than are murine EC cells. Murine EC-like cells derived from embryos in oitro are also available6,7; however, culture conditions for these cells can be more demanding than those required for conventional EC cell lines, and it is likely that the response to retinoids is the same. The methods described here are generally applicable to conventional murine EC lines. Many such lines are documented. 2 As mentioned above, the cells show widely differing propensities for differentiation in tumor form; however, cells from most EC lines differentiate readily in response to all-trans-retinoic acid (RA) (except for mutant or variant lines selected for lack of retinoid responsiveness). Cells with a high propensity for differentiation, and which can give rise to a variety of cell types in tumors or in culture, are particularly useful for studying patterns of differentiation. As a rule, such cultures are less likely to contain foci of cells refractory to retinoid action (see below). On the other hand, monitoring differentiation in these cultures can be complicated by the different phenotypes of the cells produced. Therefore, in experiments to evaluate the potencies of various retinoids, EC cell lines with more restricted differentiation profiles are generally easier to assess. The culture of murine EC cells can be carried out in any laboratory equipped with standard tissue culture equipment (laminar flow hood, inverted microscope, humidified CO2 incubator, etc.). Because of their rapid growth rate (doubling time 10-14 hr), EC cells can be readily expanded for many experimental procedures involving protein and nucleic acid isolation. Embryonal carcinoma cells are not contact-inhibited; they pack tightly in monolayer cultures, giving very high final densities. The cells are also capable of growing clonally in semisolid medium. 6 G. R. Martin and L. F. Lock, in "Teratocarcinoma Stem Cells" (L. M. Silver, G. R. Martin, and S. Strickland, eds.), p. 635. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 7 E. J. Robertson, M. H. Kaufman, A. Bradley, and M. J. Evans, in "Teratocarcinoma Stem Ceils" (L. M. Silver, G. R. Martin, and S. Strickland, eds.), p. 647. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983.
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Materials Dulbecco's modified Eagle's medium (DMEM), 1 g/liter glucose (Gibco, Grand Island, NY) Fetal calf serum (FCS), heat-inactivated at 55 ° for 20 min and stored at - 2 0 ° (Gibco) Ca 2+- and Mg2+-free phosphate-buffered saline (PBS) (Gibco) 0. I% Gelatin (swine skin, Type I; Sigma, St. Louis, MO) dissolved in double-distilled water by mild heating; while still warm, the solution is sterile-filtered through a 0.45-/~m Nalgene tissue culturegrade disposable filter unit, then stored at 4 ° Trypsin-EDTA solution (0.05% Trypsin, 0.53 mM EDTA) (Gibco), dispensed into aliquots and stored frozen at - 2 0 ° Penicillin-streptomycin solution (10,000 U/ml penicillin G, 10 mg/ml streptomycin), stored frozen at - 2 0 ° (Gibco) Kanamycin solution (10 mg/ml), stored frozen at - 2 0 ° (Gibco) Retinoids (e.g., RA) are dissolved in ethanol (final concentration - 10 mM); RA is stored as a stock solution for a maximum of 2 weeks in the dark at - 7 0 °, whereas other retinoids might be less soluble in ethanol than RA and/or more stable when stored at - 7 0 ° Methods Since many retinoids are light-sensitive, it is desirable to carry out all tissue culture operations in a room with amber lighting. Embryonal carcinoma cells are grown in DMEM supplemented with fresh glutamine (4 mM) and 10% FCS (S-DMEM) at 37° in a humidified atmosphere of 5% CO2 in air. The use of antibiotics is optional. Working concentrations of antibiotics are I00 U/ml penicillin, 100/.~g/ml streptomycin, and/or 100 /zg/ml kanamycin. Although EC cells grow well in the low concentrations of glucose present in S-DMEM, higher concentrations (e.g., 4.5 g/liter) will result in accelerated growth. The drawback of using the high glucose formulation is that the medium becomes acidic very quickly, and this can lead to death of the cultures, particularly when they reach high densities. Thus, if large numbers of cells are required, the cultures can be grown in high glucose, but the medium will have to be changed at least every other day and the cells will have to be passaged on a regular basis, for example, every second or third day at a split ratio of 1 : 5 to 1 : 20. For subculturing, cells are rinsed once with PBS and incubated for 2-3 min at room temperature with enough trypsin-EDTA solution to cover the cells (normally 0.5 ml/25 cm z flask). Some EC cell lines are more difficult to remove from the substratum and require longer incubation or incubation at 37 °. In some instances gentle shaking of the tissue culture
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vessel will help to detach the cells from the plastic. However, EC cells are very sensitive to trypsin-EDTA and will lyse if treated too vigorously or for too long. To ensure good recovery of viable cells, trypsin digestion can be monitored under an inverted microscope. Trypsin is inactivated by adding prewarmed S-DMEM. A single-cell suspension is usually obtained by gently pipetting the mixture several times. Embryonal carcinoma cells from some lines tend to be dislodged from the substratum as small aggregates rather than single cells. For routine subculturing this does not present a problem. However, if cell counting is to be performed, the potential for experimental error is too great to be ignored. We have found that by gently passing the suspension through a 19-gauge needle, with care to minimize bubbling, greater than 98% of the aggregates can be dispersed into single cells with little or no noticeable effect on cell viability. The cells should be allowed to settle on, and attach to, the substratum for at least 6 hr before any additions are made to the medium. Some EC cells (e.g., F9, Nulli-SCC 1) adhere very poorly to tissue culture vessels. A gelatin coating can overcome this problem: at least 10 rain prior to seeding the cells, enough 0.1% gelatin solution is added to the vessel to completely cover the surface. After standing at room temperature, the gelatin solution is aspirated and the vessel rinsed once with PBS before medium is added. When aggregate formation is desired, cells are plated in bacteriological grade dishes. 3 Induction of Differentiation To induce differentiation of cells in monolayers or aggregates with RA, a 10 m M solution in ethanol is diluted at least 100-fold in S-DMEM, and appropriate aliquots are added to cells to give final concentrations between 1 nM and 1/zM. For RA and most other retinoids, lower concentrations do not produce a noticeable effect, and higher concentrations can be toxic. (If serum-free medium is used, the retinoid concentration will have to be lowered dramatically or toxicity will result.) The final ethanol concentration should be less than 0.5%. For long-term cultures, medium containing RA is replaced at least every 48 hr. Since EC cells can metabolize RA and certain other retinoids very effectively (see Ref. 8), in experiments where continuous presence of retinoid is critical, RA should be added every 24 hr. Although not an inducer of differentiation by itself, 1 mM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) can be added to enhance the differentiation-inducing potency of retinoids on at least some EC c e l l s . 4 8 M. L. Gubler and M. I; Sherman, this volume [60].
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Comments
There are several factors that the investigator should consider when designing an experimental protocol with EC cells. For example, after retinoids are added to EC cells, cell proliferation will be dramatically curtailed beyond 24 hr as differentiation occurs. Some cell death is also likely. Therefore, if differentiation is extensive or complete at the end of the experiment, there might only be two or three population doublings from the time of retinoid addition. Another important consideration is the existence in most EC cell cultures of a small proportion of cells which will be at least transiently refractory to retinoid-induced differentiation. (This phenomenon is particularly marked in cell lines such as NulIi-SCC1 or F9 which generally differentiate less readily than pluripotent lines, but the extent of retinoid refractoriness should be evaluated empirically whenever a cell line is used for the first time.) The result is the emergence with time of colonies of EC-like cells against a background of fiat and enlarged differentiated derivatives. 9 This is most evident when cells are induced to differentiate at high density. Thus, with some EC lines, if cultures are treated with retinoids when they are relatively dense, cells which are refractory to retinoid action can eventually overgrow differentiated cells, and this could lead to inappropriate interpretations of experimental results. It is, therefore, desirable in long-term experiments to plate cells at low density and, if large amounts of cells are required, either to use larger culture dishes (e.g., Nunc 245 x 245 x 20 mm plates; Inter Med, Roskilde, Denmark) or to increase the number of dishes per experiment. This need not necessarily require significant scaling up of subsequent biochemical procedures: for example, total RNA can be extracted by the guanidinium thiocyanate procedure from as many as 10 large Nunc plates by merely doubling the amount of solution routinely used for a single plate (see below), which is then transferred sequentially from plate to plate. Assessment of Embryonal Carcinoma Cell Differentiation It is not within the scope of this chapter to review all of the alterations that accompany differentiation of EC cells. Instead, we concentrate on some consistent changes exhibited by EC cells that have been widely used as convenient indicators of differentiation. Generally, these can be divided into disappearance of properties characteristic of undifferentiated EC cells (EC cell markers) and appearance of properties shared by many or most of the cell types that result from retinoid-induced differentiation of EC cells in vitro (differentiation markers). 9 M. I. Sherman, K. I. Matthaei, and J. Schindler, Ann. N.Y. Acad. Sci. 359, 192 (1981).
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Embryonal Carcinoma Cell Markers Morphology. Although there are differences in the morphology of EC cells from various lines and a trained investigator can distinguish cells from different lines, EC cells are commonly small with large nucleus/ cytoplasm ratios and prominent nucleoli. The cells typically (although not always) grow in tightly packed clusters, and because they fail to produce an abundant extracellular matrix, cellular boundaries within clusters are generally indistinct. When EC cells differentiate, they can assume a variety of phenotypes, but in general the cells are larger and flatter with reduced nucleus/cytoplasm ratios and more discretely defined cell boundaries. Morphology is a convenient way to monitor differentiation qualitatively as an experiment progresses. However, there is a great danger in using morphological change as a sine qua non for differentiation as retinoids and other chemical inducers can alter morphology without inducing overt cell differentiation. ~0Note: Conclusions about differentiation of EC cells should never be based exclusively on cell morphology. In fact, it is prudent to use two or more markers when evaluating the differentiation status of cells in culture. Growth Properties. Two other features of EC cells that are consistently and dramatically reduced or extinguished as a result of differentiation are colony-forming efficiency (CFE) in liquid culture H and anchorage-independent growth in semisolid medium. 12 Such reductions might not necessarily be indicative of differentiation: cytotoxic agents would obviously give a similar result. However, this eventuality can be eliminated by toxicity testing of the retinoid at effective concentrations against EC cells at nonclonal densities in liquid medium. With this caveat, these tests can be reliably used not only to signal cell differentiation, but also to indicate the time at which cells become irreversibly committed to differentiate (i.e., they go on to generate differentiated cells even after the inducer is removed). 11In fact, a convenient way to assay for these properties is in the same way that commitment studies are carried out: cells treated with retinoid in S-DMEM for at least 48 hr (a period adequate for commitment to differentiation to occur in the great majority of cells") are washed extensively with PBS to remove retinoid and treated with trypsin-EDTA as described above. Fresh S-DMEM is added and the cells are counted. Cells are plated in liquid or semisolid medium (the latter contains a 1 : 1 mixture of 2× S-DMEM and 1.92% methylcellulose) at l0 M. I. Sherman, M. A. Eglitis, and R. Thomas, J. Embryol. Exp. Morphol. 93, 179 (1986). 11 M. I. Sherman, M. L. Gubler, U. Barkai, M. I. Harper, G. Coppola, and J. Yuan, Ciba Found. Symp. 113, 42 (1985). 12 S.-Y. Wang and L. Gudas, J. Biol. Chem. 2,59, 5899 (1984).
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densities low enough to allow accurate counting of colonies (e.g., 200 or fewer colonies/60-mm-diameter culture dish). It is desirable to plate triplicate dishes for each retinoid-treated sample. Cultures should be incubated for at least 7 days in liquid medium and for as many as 14 days in semisolid medium. At the end of the culture period, colonies are counted after fixing and staining with 0.2% crystal violet (liquid culture) or 0.05% iodonitrotetrazolium (methylcellulose). An inverse relationship is to be expected between number of colonies and EC cell differentiation. SSEA-1. One of the most useful antigenic markers for following differentiation of EC cells is the surface antigen SSEA-1 (stage-specific embryonic antigen-I). 13This surface marker is expressed on most or all EC cells but is rapidly lost when the cells differentiate, regardless of the inducer added or direction of differentiation. Expression of this marker can be easily assessed by indirect immunofluorescence using a mouse monoclonal antibody developed by Solter and Knowles.13 Standard protocols for indirect immunofluorescence work reasonably well with most EC cells. However, cells from some EC lines do not attach firmly to glass coverslips and are lost during washing. With such cells the procedure can be carried out in gelatin-coated culture dishes (gelatin coating of glass coverslips does not markedly improve EC cell adhesion). For the assay, retinoid-treated cells are rinsed twice with PBS and fixed for 10 min with cold methanol or paraformaldehyde. After two rinses with PBS, a small circle (0.5-1 cm in diameter) is etched in the center of the dish with a scalpel. The area immediately outside the circle is dried with filter paper. Immunostaining is then performed by incubating the cells inside the etched circle with 50 to 70/~1 antibody, appropriately diluted. After washing and exposure to fluorescein-labeled antiimmunoglobulin, a drop of glycerol/PBS (9: 1, v/v) is placed in the circle and covered with a coverslip for microscopic analysis. Viral Gene Expression. Another common property shared by EC cells is that they are nonpermissive to expression of viral gene products following infection with papovaviruses and type-C retroviruses.14 Although the mechanisms by which EC cells restrict viral gene expression are not yet fully understood, it is known that following differentiation the block to virus expression is removed. This attribute has been exploited to evaluate the differentiation status of cultured EC cells. Treated or untreated EC cells are infected at high multiplicity with either polyoma or SV40 virus. At appropriate times (see below), cells are assayed for T-antigen expres13 D. Solter and B. B. Knowles, Proc. Natl. Acad. Sci. U.S.A. 75, 5565 (1978). ~4S. Astigiano, M. I. Sherman, and P. Abarzua, Environ. Health Perspect. 80, 25 (1989).
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sion by indirect immunofluorescence. The procedure is essentially as described for SSEA-1 antigen analysis. Positive cells are identified by strong and specific nuclear (but not nucleolar) staining. The results can be quantitated by analyzing several randomly chosen fields and scoring positive nuclei among a total of, for example, 1000 cells. Maximum expression occurs 3 to 4 days after infection, but significant immunostaining can be detected as early as 24 hr postinfection. Infected mouse 3T3 cells and uninfected EC cells should be used as positive and negative controls, respectively, in these experiments. More than 70% of SV40-infected 3T3 cells should exhibit T-antigen staining. Infected but untreated EC cells should be uniformly negative, and, even in fully differentiated cultures, the fraction of T-antigen-positive cells will be much smaller than that observed for 3T3 fibroblasts. Differentiation Markers The fact that EC cells can differentiate into a large variety of cell types implies that a variety of markers can be used to assess and monitor EC cell differentiation. The choice of markers to monitor, therefore, will depend in large part on the cell line being analyzed and the type of inducer.
Plasminogen Activator Plasminogen activator was one of the first criteria used to monitor differentiation of EC cells. 15Very low levels of plasminogen activator are produced and secreted by EC cells. Synthesis and secretion of this enzyme are enhanced by as much as two orders of magnitude upon differentiation of EC cells into endodermlike cells, particularly parietal endoderm (e.g., F9 cells treated with RA plus dbcAMP or micromolar levels of RA alone). Even when cells differentiate to other phenotypes there appears to be a transient (within 48 hr) but significant increase in plasminogen activator secretion. For these reasons this differentiation marker is useful for following retinoid-induced differentiation in early stages. Plasminogen activator is also a very sensitive parameter since elevated secretion of this enzyme by only a small percentage of cells in the culture can be readily detected. A quantitative assay has been used to measure secretion of plasminogen activator by EC cells. 16This assay measures plasminogen-dependent fibrinolysis of 125I-labeled fibrinogen. Cells are cultured and induced to t5 M. I. Sherman, S. Strickland, and E. Reich, Cancer Res. 36, 4208 (1976). 16 S. Strickland, E. Reich, and M. I. Sherman, Cell (Cambridge, Mass.) 9, 231 (1976).
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differentiate as described above. Twenty-four hours before a time point is to be taken, medium is removed, cells are rinsed thoroughly with PBS, and S-DMEM medium containing heat-inactivated, plasminogen-depleted serum 17 is added. After incubation, conditioned medium is collected and stored at - 2 0 ° and cells are counted. For the assay, a mixture of 1251labeled fibrinogen and unlabeled fibrinogen (30/zg, -4000 cpm//xg) in a 1 : 11 mixture of PBS/water is added to each well of 24-well plates and allowed to dry at 37° . Dried plates can be stored for several days at room temperature. Two hours prior to assay, 0.3 ml of a 10% solution of plasminogendepleted serum in 0.1 M Tris-HC1, pH 7.4, is added to each well. After a 2-hr incubation at 37°, each well is washed twice with 0.1 M Tris-HC1, pH 8.1. For measuring plasminogen activator secretion, 20/xl conditioned medium and 5 ttl plasminogen (10/zg) in 225 ~1 of 0.1 M Tris-HCl, pH 8.1, are added to each well and incubated for 2 hr at 37°. Reaction mixtures are collected, each well is washed with 250 /xl Tris buffer, and the wash solution is pooled with the reaction mixture. Samples are then counted in a 3' counter to determine the release of 1251. Samples should be run in triplicate. Two controls should always be included: in one, wells are incubated with 25/zl trypsin-EDTA solution to determine the total number of 1251counts per minute that can be released with protease; a second control consists of medium previously incubated without cells (also for 24 hr at 37°), to determine background levels of soluble 1251.When characterizing a new cell line, it is also prudent to include control samples lacking plasminogen to ensure that all fibrinolysis is plasmin-dependent. For quantitation, the solubilized counts in the control medium samples are subtracted from experimental values, and the results are normalized for trypsin-degradable 125I-labeled fibrin and expressed on a cell number basis. Plasminogen activator can also be qualitatively determined in situ by the fibrin-agar assay as outlined by Sherman et al. 15 Structural Proteins
There are several cell surface and cytoskeletal proteins for which antibodies are available. These proteins can be easily detected by indirect immunostaining. Fibronectin is synthesized by EC cells but is not retained in association with the cell surface. On cell differentiation into parietal endoderm, fibronectin can accumulate as part of the basement membrane.18 It also becomes surface-associated when fibroblastlike cells i7 L. Ossowski, J. C. Unkeless, A. Tobia, J. P. Quigley, D. B. Rifkin, and E. Reich, J. Exp. Med. 137, 112 (1973). 18 j. Wolfe, V. Mautner, B. Hogan, and R. Tilly, Exp. Cell Res. 118, 63 (1979).
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are formed. Increased synthesis and extracellular matrix deposition of three other basement membrane proteins, namely, collagen IV, laminin, and entactin, can be detected when EC cells differentiate into endodermlike cells) 9,2° On the other hand, a-fetoprotein antibodies are useful for staining visceral endodermlike cells. 21 These latter markers might be of more limited utility, depending on patterns of differentiation of the EC lines under study. Cytoskeletal proteins have also been employed to evaluate EC cell differentiation, although such markers are perhaps more useful as secondary indicators. Keratin intermediate filaments are detected by immunofluorescence when EC cells differentiate into epithelial cells. 22 Monoclonal antibodies TROMA-1 and TROMA-3 react with different intermediate filament components present in gut and fetal liver as well as mesoderm and endoderm-derived epithelia in adult animals. Both fail to react with EC cells, but they detect an ordered intermediate filament network present in differentiated cells: TROMA-1 reacts strongly with both visceral and parietal endoderm cells, whereas TROMA-3 is more specific, reacting only with parietal endoderm. 23 Two other intermediate filament components not expressed in EC cells, Endo A and Endo B, are readily detectable in trophoblast, visceral endoderm, parietal endoderm, and liver cells but not in myoblasts, neuroblastoma cells, or keratinocytes. 24 Differentiation of EC cells into glial-like cells and myoblastlike cells is accompanied by neurofilament and desmin expression, respectively. 25 mRNA Analyses cDNA probe technology can be utilized to evaluate EC cell differentiation. These probes can often be used to study differentiation at the molecular level before measurable morphological or biochemical changes occur. Several cDNA clones for genes either up- or downregulated have been reported, some representing structural proteins or enzymes previously known to be regulated, as described above (e.g., collagen IV, 19 A. Grover and E. D. Adamson, J. Biol. Chem. 260, 12252 (1985). 2o A. R. Cooper, A. Taylor, and B. L. M. Hogan, Dev. Biol. 99, 510 (1983). 2~ B. L. M. Hogan, A. Taylor, and E. Adamson, Nature (London) 291, 235 (1981). 22 D. Paalin, H. Jakob, F. Jacob, K. Weber, and M. Osborn, Differentiation 22, 90 (1982). 23 R. Kemler, P. Brulet, M. T. Schnebeley, J. Gaillard, and F. Jacob, J. Embryol. Exp. Morphol. 64, 45 (1981). 24 A. Grover, R. Oshima, and E. D. Adamson, ]. Cell Biol. 96, 1690 (1983). 25 E. M. V. Jones-Villeneuve, M. W. McBurney, K. A. Rogers, and V. 1. Kalnins, J. Cell Biol. 94, 253 (1982).
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laminin, entactin, a-fetoprotein), 26-28 others encoding novel proteins of u n k n o w n function. 27,29
For R N A isolation from EC cells, we have found a slightly modified version of the procedure described by Chomczynski and Sacchi 3° to be particularly useful and convenient. Because of its simplicity, it allows simultaneous processing of a large number of samples, as would be required, for example, in time-course experiments. Embryonal carcinoma cells are plated as described above (preferentially in culture dishes rather than flasks). At the end of the incubation period, cells are washed with PBS and lysed in situ by the addition of 2 ml/100-mm dish of guanidine thiocyanate solution (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). The lysate is then transferred with the help of a rubber policeman either to a siliconized and baked Corex 15-ml glass tube or to a disposable 14-ml polypropylene tube (Falcon 2059). The R N A is extracted by adding 0.2 ml of 2 M sodium acetate, pH 4.0, 2 ml water-saturated phenol, and 0.4 ml chloroform. After vortexing for 10 sec, the suspension is cooled on ice for 15 min and centrifuged at 8000 rpm (e.g., Sorval SS-34 rotor) for 20 rain at 4°. The aqueous phase containing the R N A is transferred to a new tube, mixed with 1 ml ice-cold 2propanol, and incubated at - 2 0 ° to precipitate the RNA. Care should be taken to leave behind the interphase, where DNA is present. The RNA is pelleted by centrifugation, resuspended in 0.3 ml guanidine thiocyanate solution, transferred to a 1.5-ml microcentrifuge tube and precipitated again with ! volume ice-cold 2-propanol or 2 volumes ice-cold ethanol. The RNA pellet is collected by centrifugation for l0 min in a microcentrifuge, washed once with 70% ice-cold ethanol, vacuum-dried, and dissolved in 50-100/xl water or 0.5% sodium dodecyl sulfate. If undissolved material is present at this point, the solution is centrifuged for 5 min in a microcentrifuge to pellet it, and the RNA can be further purified by ethanol precipitation from 0.3 M sodium acetate. Yields from a 100-mm dish vary from 100 to 200/xg of total RNA depending on cell density. The OD260/OD280 ratio in l0 m M Tris-HCl, pH 7.5, is normally not less than 2. RNA purified by this procedure is undegraded and stable for many months when stored at - 2 0 ° and can be used for cDNA synthesis, 26 S.-Y. Wang and L. Gudas, Proc. Natl. Acad. Sci. U.S.A. 80, 5880 (1983). 27 S.-Y. Wang, G. J. LaRosa, and L. J. Gudas, Dev. Biol. 107, 75 (1985). 28 p. R. Young and S. M. Tilghman, Mol. Cell. Biol. 4, 898 (1984). 29 R. A. Levine, G. J. La Rosa, and L. J. Gudas, Mol. Cell. Biol. 4, 2142 (1984). 30 p. Chomczynski and N. Sacchi, Anal. Biochern. 162, 156 (1987).
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Northern blots, poly(A) +, selection and S1 protection assays (see, e.g., Ref. 31). 31 j. F. Grippo and M. I. Sherman, this series, Vol. 190 [16].
[36] P a r a s i t e R e t i n o i d - B i n d i n g P r o t e i n s By BRAHMA P. SANI Introduction The major theory to explain the molecular mechanism of retinoid action in the control of epithelial differentiation and growth centers on the well-known cellular binding proteins for retinol and retinoic acid, CRBP and CRABP, respectively, 1-4 and on the recently discovered nuclear receptors for retinoic acid (RARs). 5-7 The role of retinoids in parasites is largely unknown although the occurrence and uptake of retinol and retinoic acid, as well as the formation of retinol from r-carotene by parasitic helminths, have been described by several investigators. Growth of some developmental stages of helminth parasites may depend on host levels of retinol. 8,9 Sturchler et al. 1o showed that the retinol concentration in adult O n c h o c e r c a v o l v u l u s was 8 times higher than that of the surrounding host tissues. Although the exact function of retinoids in parasites is not clear, it may be assumed, by analogy to host tissues, that they control certain vital biological functions of the parasites, such as differentiation, growth, and reproduction. In our efforts to study the interactions of retinoids with parasitic components, we discovered and partially characterized specific
I M. M. Bashor, D. O. Toft, and F. Chytil, Proc. Natl. Acad. Sci. U.S.A. 70, 3483 (1973). 2 B. P. Sani and D. L. Hill, Biochem. Biophys. Res. Commun. 61, 1276 (1974). 3 D. E. Ong and F. Chytil, J. Biol. Chem. 250, 6113 (1975). 4 B. P. Sani and D. L. Hill, Cancer Res. 36, 409 (1976). 5 M. Petkovich, N. J. Brand, A. Krust, and P. Chambon, Nature (London) 330, 444 (1987). 6 V. Giguere, E. S. Ong, P. Sequi, and R. M. Evans, Nature (London) 330, 624 (1987). 7 N. Brand, M. Petkovich, A. Krust, P. Chambon, H. Dethe, A. Marchio, P. Tiollais, and A. Dejean, Nature (London) 332, 850 (1988). 8 D. Mahalanabis, K. N. Jalan, T. K. Maitra, and S. R. Agarwal Am. J. Clin. Nutr. 29, 1372 (1976). 9 D. M. Storey, Z. Parasitenkd. 67, 309 (1982). 10 D. Sturchler, B. Holzer, A. Hanck, and A. Oegremont, Acta Trop. 40, 261 (1983).
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[35] D i f f e r e n t i a t i o n o f E m b r y o n a l C a r c i n o m a Cells in R e s p o n s e to R e t i n o ! d s
By PATRICIO ABAaZ6A and MICHAEL I. SHERMAN Introduction Embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, offer an attractive experimental model for studying differentiation because they resemble early embryonic stem cells.l Embryonal carcinoma cells were initially isolated from primary cultures of cells derived from teratocarcinomas, and from these many clonal cell lines have been developed. The propensity for differentiation of cells from these lines can be assessed in vivo; such studies have indicated a broad spectrum of differentiation potential among the lines. 2 When so-called pluripotent EC cells (e.g., PCC3, PC-13) are injected subcutaneously into syngeneic mice, they give rise to teratocarcinomas which typically contain many differentiated cell types that can represent all three primary germ layers. At the other extreme, so-called nullipotent EC cells (e.g., Nulli-SCC1, F9) show little or no histological evidence of differentiation. In fact, this classification is artificial insofar as there is probably a continuum of potential for differentiation in tumors if one considers large numbers of EC lines. In tissue culture, the ability of EC cells to differentiate also varies markedly. Cells from most EC lines remain undifferentiated or differentiate at low frequency when growing exponentially. Some lines differentiate readily on reaching confluence. When EC cells are prevented from attaching to the substratum, they form multicellular aggregates, and under these conditions cells from some of the lines undergo differentiation. 3 A variety of chemicals have been shown to induce EC cell differentiation; retinoids are among the most potent chemical inducers of EC cell differentiation, with effective concentrations typically in the nanomolar range. Even EC cells such as NulIi-SCC1 and F9, which differentiate poorly in tumors, do so readily in culture in response to retinoids. 4,5 Because the G. R. Martin, Science 2119, 768 (1980). 2 G. R. Martin, in "Teratocarcinoma Stem Cells" (L. M. Silver, G. R. Martin, and S. Strickland, eds.), p. 690. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 3 M. I. Sherman, in "Teratomas and Differentiation" (M. I. Sherman and D. Solter, eds.), p. 189. Academic Press, New York, 1975. 4 S. Strickland and V. Mahdavi, Cell (Cambridge, Mass.) 15, 393 (1978). 5 A. M. Jetten, M. E. R. Jetten, and M. I. Sherman, Exp. Cell Res. 124, 381 (1979).
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mechanisms by which retinoids induce differentiation of EC cells might be relevant to their biochemical and biological effects on a large variety of cell types, and because differentiation is easily monitored in EC cells, this cell system is useful for studying retinoid action. We describe below techniques for the culture of EC cells from established lines and for evaluating the differentiation of these cells in response to retinoids. Culture of Embryonal Carcinoma Cells Although human EC cells have been cultured, 2 as a rule they are less responsive to retinoids than are murine EC cells. Murine EC-like cells derived from embryos in oitro are also available6,7; however, culture conditions for these cells can be more demanding than those required for conventional EC cell lines, and it is likely that the response to retinoids is the same. The methods described here are generally applicable to conventional murine EC lines. Many such lines are documented. 2 As mentioned above, the cells show widely differing propensities for differentiation in tumor form; however, cells from most EC lines differentiate readily in response to all-trans-retinoic acid (RA) (except for mutant or variant lines selected for lack of retinoid responsiveness). Cells with a high propensity for differentiation, and which can give rise to a variety of cell types in tumors or in culture, are particularly useful for studying patterns of differentiation. As a rule, such cultures are less likely to contain foci of cells refractory to retinoid action (see below). On the other hand, monitoring differentiation in these cultures can be complicated by the different phenotypes of the cells produced. Therefore, in experiments to evaluate the potencies of various retinoids, EC cell lines with more restricted differentiation profiles are generally easier to assess. The culture of murine EC cells can be carried out in any laboratory equipped with standard tissue culture equipment (laminar flow hood, inverted microscope, humidified CO2 incubator, etc.). Because of their rapid growth rate (doubling time 10-14 hr), EC cells can be readily expanded for many experimental procedures involving protein and nucleic acid isolation. Embryonal carcinoma cells are not contact-inhibited; they pack tightly in monolayer cultures, giving very high final densities. The cells are also capable of growing clonally in semisolid medium. 6 G. R. Martin and L. F. Lock, in "Teratocarcinoma Stem Cells" (L. M. Silver, G. R. Martin, and S. Strickland, eds.), p. 635. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983. 7 E. J. Robertson, M. H. Kaufman, A. Bradley, and M. J. Evans, in "Teratocarcinoma Stem Ceils" (L. M. Silver, G. R. Martin, and S. Strickland, eds.), p. 647. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1983.
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Materials Dulbecco's modified Eagle's medium (DMEM), 1 g/liter glucose (Gibco, Grand Island, NY) Fetal calf serum (FCS), heat-inactivated at 55 ° for 20 min and stored at - 2 0 ° (Gibco) Ca 2+- and Mg2+-free phosphate-buffered saline (PBS) (Gibco) 0. I% Gelatin (swine skin, Type I; Sigma, St. Louis, MO) dissolved in double-distilled water by mild heating; while still warm, the solution is sterile-filtered through a 0.45-/~m Nalgene tissue culturegrade disposable filter unit, then stored at 4 ° Trypsin-EDTA solution (0.05% Trypsin, 0.53 mM EDTA) (Gibco), dispensed into aliquots and stored frozen at - 2 0 ° Penicillin-streptomycin solution (10,000 U/ml penicillin G, 10 mg/ml streptomycin), stored frozen at - 2 0 ° (Gibco) Kanamycin solution (10 mg/ml), stored frozen at - 2 0 ° (Gibco) Retinoids (e.g., RA) are dissolved in ethanol (final concentration - 10 mM); RA is stored as a stock solution for a maximum of 2 weeks in the dark at - 7 0 °, whereas other retinoids might be less soluble in ethanol than RA and/or more stable when stored at - 7 0 ° Methods Since many retinoids are light-sensitive, it is desirable to carry out all tissue culture operations in a room with amber lighting. Embryonal carcinoma cells are grown in DMEM supplemented with fresh glutamine (4 mM) and 10% FCS (S-DMEM) at 37° in a humidified atmosphere of 5% CO2 in air. The use of antibiotics is optional. Working concentrations of antibiotics are I00 U/ml penicillin, 100/.~g/ml streptomycin, and/or 100 /zg/ml kanamycin. Although EC cells grow well in the low concentrations of glucose present in S-DMEM, higher concentrations (e.g., 4.5 g/liter) will result in accelerated growth. The drawback of using the high glucose formulation is that the medium becomes acidic very quickly, and this can lead to death of the cultures, particularly when they reach high densities. Thus, if large numbers of cells are required, the cultures can be grown in high glucose, but the medium will have to be changed at least every other day and the cells will have to be passaged on a regular basis, for example, every second or third day at a split ratio of 1 : 5 to 1 : 20. For subculturing, cells are rinsed once with PBS and incubated for 2-3 min at room temperature with enough trypsin-EDTA solution to cover the cells (normally 0.5 ml/25 cm z flask). Some EC cell lines are more difficult to remove from the substratum and require longer incubation or incubation at 37 °. In some instances gentle shaking of the tissue culture
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vessel will help to detach the cells from the plastic. However, EC cells are very sensitive to trypsin-EDTA and will lyse if treated too vigorously or for too long. To ensure good recovery of viable cells, trypsin digestion can be monitored under an inverted microscope. Trypsin is inactivated by adding prewarmed S-DMEM. A single-cell suspension is usually obtained by gently pipetting the mixture several times. Embryonal carcinoma cells from some lines tend to be dislodged from the substratum as small aggregates rather than single cells. For routine subculturing this does not present a problem. However, if cell counting is to be performed, the potential for experimental error is too great to be ignored. We have found that by gently passing the suspension through a 19-gauge needle, with care to minimize bubbling, greater than 98% of the aggregates can be dispersed into single cells with little or no noticeable effect on cell viability. The cells should be allowed to settle on, and attach to, the substratum for at least 6 hr before any additions are made to the medium. Some EC cells (e.g., F9, Nulli-SCC 1) adhere very poorly to tissue culture vessels. A gelatin coating can overcome this problem: at least 10 rain prior to seeding the cells, enough 0.1% gelatin solution is added to the vessel to completely cover the surface. After standing at room temperature, the gelatin solution is aspirated and the vessel rinsed once with PBS before medium is added. When aggregate formation is desired, cells are plated in bacteriological grade dishes. 3 Induction of Differentiation To induce differentiation of cells in monolayers or aggregates with RA, a 10 m M solution in ethanol is diluted at least 100-fold in S-DMEM, and appropriate aliquots are added to cells to give final concentrations between 1 nM and 1/zM. For RA and most other retinoids, lower concentrations do not produce a noticeable effect, and higher concentrations can be toxic. (If serum-free medium is used, the retinoid concentration will have to be lowered dramatically or toxicity will result.) The final ethanol concentration should be less than 0.5%. For long-term cultures, medium containing RA is replaced at least every 48 hr. Since EC cells can metabolize RA and certain other retinoids very effectively (see Ref. 8), in experiments where continuous presence of retinoid is critical, RA should be added every 24 hr. Although not an inducer of differentiation by itself, 1 mM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) can be added to enhance the differentiation-inducing potency of retinoids on at least some EC c e l l s . 4 8 M. L. Gubler and M. I; Sherman, this volume [60].
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Comments
There are several factors that the investigator should consider when designing an experimental protocol with EC cells. For example, after retinoids are added to EC cells, cell proliferation will be dramatically curtailed beyond 24 hr as differentiation occurs. Some cell death is also likely. Therefore, if differentiation is extensive or complete at the end of the experiment, there might only be two or three population doublings from the time of retinoid addition. Another important consideration is the existence in most EC cell cultures of a small proportion of cells which will be at least transiently refractory to retinoid-induced differentiation. (This phenomenon is particularly marked in cell lines such as NulIi-SCC1 or F9 which generally differentiate less readily than pluripotent lines, but the extent of retinoid refractoriness should be evaluated empirically whenever a cell line is used for the first time.) The result is the emergence with time of colonies of EC-like cells against a background of fiat and enlarged differentiated derivatives. 9 This is most evident when cells are induced to differentiate at high density. Thus, with some EC lines, if cultures are treated with retinoids when they are relatively dense, cells which are refractory to retinoid action can eventually overgrow differentiated cells, and this could lead to inappropriate interpretations of experimental results. It is, therefore, desirable in long-term experiments to plate cells at low density and, if large amounts of cells are required, either to use larger culture dishes (e.g., Nunc 245 x 245 x 20 mm plates; Inter Med, Roskilde, Denmark) or to increase the number of dishes per experiment. This need not necessarily require significant scaling up of subsequent biochemical procedures: for example, total RNA can be extracted by the guanidinium thiocyanate procedure from as many as 10 large Nunc plates by merely doubling the amount of solution routinely used for a single plate (see below), which is then transferred sequentially from plate to plate. Assessment of Embryonal Carcinoma Cell Differentiation It is not within the scope of this chapter to review all of the alterations that accompany differentiation of EC cells. Instead, we concentrate on some consistent changes exhibited by EC cells that have been widely used as convenient indicators of differentiation. Generally, these can be divided into disappearance of properties characteristic of undifferentiated EC cells (EC cell markers) and appearance of properties shared by many or most of the cell types that result from retinoid-induced differentiation of EC cells in vitro (differentiation markers). 9 M. I. Sherman, K. I. Matthaei, and J. Schindler, Ann. N.Y. Acad. Sci. 359, 192 (1981).
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Embryonal Carcinoma Cell Markers Morphology. Although there are differences in the morphology of EC cells from various lines and a trained investigator can distinguish cells from different lines, EC cells are commonly small with large nucleus/ cytoplasm ratios and prominent nucleoli. The cells typically (although not always) grow in tightly packed clusters, and because they fail to produce an abundant extracellular matrix, cellular boundaries within clusters are generally indistinct. When EC cells differentiate, they can assume a variety of phenotypes, but in general the cells are larger and flatter with reduced nucleus/cytoplasm ratios and more discretely defined cell boundaries. Morphology is a convenient way to monitor differentiation qualitatively as an experiment progresses. However, there is a great danger in using morphological change as a sine qua non for differentiation as retinoids and other chemical inducers can alter morphology without inducing overt cell differentiation. ~0Note: Conclusions about differentiation of EC cells should never be based exclusively on cell morphology. In fact, it is prudent to use two or more markers when evaluating the differentiation status of cells in culture. Growth Properties. Two other features of EC cells that are consistently and dramatically reduced or extinguished as a result of differentiation are colony-forming efficiency (CFE) in liquid culture H and anchorage-independent growth in semisolid medium. 12 Such reductions might not necessarily be indicative of differentiation: cytotoxic agents would obviously give a similar result. However, this eventuality can be eliminated by toxicity testing of the retinoid at effective concentrations against EC cells at nonclonal densities in liquid medium. With this caveat, these tests can be reliably used not only to signal cell differentiation, but also to indicate the time at which cells become irreversibly committed to differentiate (i.e., they go on to generate differentiated cells even after the inducer is removed). 11In fact, a convenient way to assay for these properties is in the same way that commitment studies are carried out: cells treated with retinoid in S-DMEM for at least 48 hr (a period adequate for commitment to differentiation to occur in the great majority of cells") are washed extensively with PBS to remove retinoid and treated with trypsin-EDTA as described above. Fresh S-DMEM is added and the cells are counted. Cells are plated in liquid or semisolid medium (the latter contains a 1 : 1 mixture of 2× S-DMEM and 1.92% methylcellulose) at l0 M. I. Sherman, M. A. Eglitis, and R. Thomas, J. Embryol. Exp. Morphol. 93, 179 (1986). 11 M. I. Sherman, M. L. Gubler, U. Barkai, M. I. Harper, G. Coppola, and J. Yuan, Ciba Found. Symp. 113, 42 (1985). 12 S.-Y. Wang and L. Gudas, J. Biol. Chem. 2,59, 5899 (1984).
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densities low enough to allow accurate counting of colonies (e.g., 200 or fewer colonies/60-mm-diameter culture dish). It is desirable to plate triplicate dishes for each retinoid-treated sample. Cultures should be incubated for at least 7 days in liquid medium and for as many as 14 days in semisolid medium. At the end of the culture period, colonies are counted after fixing and staining with 0.2% crystal violet (liquid culture) or 0.05% iodonitrotetrazolium (methylcellulose). An inverse relationship is to be expected between number of colonies and EC cell differentiation. SSEA-1. One of the most useful antigenic markers for following differentiation of EC cells is the surface antigen SSEA-1 (stage-specific embryonic antigen-I). 13This surface marker is expressed on most or all EC cells but is rapidly lost when the cells differentiate, regardless of the inducer added or direction of differentiation. Expression of this marker can be easily assessed by indirect immunofluorescence using a mouse monoclonal antibody developed by Solter and Knowles.13 Standard protocols for indirect immunofluorescence work reasonably well with most EC cells. However, cells from some EC lines do not attach firmly to glass coverslips and are lost during washing. With such cells the procedure can be carried out in gelatin-coated culture dishes (gelatin coating of glass coverslips does not markedly improve EC cell adhesion). For the assay, retinoid-treated cells are rinsed twice with PBS and fixed for 10 min with cold methanol or paraformaldehyde. After two rinses with PBS, a small circle (0.5-1 cm in diameter) is etched in the center of the dish with a scalpel. The area immediately outside the circle is dried with filter paper. Immunostaining is then performed by incubating the cells inside the etched circle with 50 to 70/~1 antibody, appropriately diluted. After washing and exposure to fluorescein-labeled antiimmunoglobulin, a drop of glycerol/PBS (9: 1, v/v) is placed in the circle and covered with a coverslip for microscopic analysis. Viral Gene Expression. Another common property shared by EC cells is that they are nonpermissive to expression of viral gene products following infection with papovaviruses and type-C retroviruses.14 Although the mechanisms by which EC cells restrict viral gene expression are not yet fully understood, it is known that following differentiation the block to virus expression is removed. This attribute has been exploited to evaluate the differentiation status of cultured EC cells. Treated or untreated EC cells are infected at high multiplicity with either polyoma or SV40 virus. At appropriate times (see below), cells are assayed for T-antigen expres13 D. Solter and B. B. Knowles, Proc. Natl. Acad. Sci. U.S.A. 75, 5565 (1978). ~4S. Astigiano, M. I. Sherman, and P. Abarzua, Environ. Health Perspect. 80, 25 (1989).
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sion by indirect immunofluorescence. The procedure is essentially as described for SSEA-1 antigen analysis. Positive cells are identified by strong and specific nuclear (but not nucleolar) staining. The results can be quantitated by analyzing several randomly chosen fields and scoring positive nuclei among a total of, for example, 1000 cells. Maximum expression occurs 3 to 4 days after infection, but significant immunostaining can be detected as early as 24 hr postinfection. Infected mouse 3T3 cells and uninfected EC cells should be used as positive and negative controls, respectively, in these experiments. More than 70% of SV40-infected 3T3 cells should exhibit T-antigen staining. Infected but untreated EC cells should be uniformly negative, and, even in fully differentiated cultures, the fraction of T-antigen-positive cells will be much smaller than that observed for 3T3 fibroblasts. Differentiation Markers The fact that EC cells can differentiate into a large variety of cell types implies that a variety of markers can be used to assess and monitor EC cell differentiation. The choice of markers to monitor, therefore, will depend in large part on the cell line being analyzed and the type of inducer.
Plasminogen Activator Plasminogen activator was one of the first criteria used to monitor differentiation of EC cells. 15Very low levels of plasminogen activator are produced and secreted by EC cells. Synthesis and secretion of this enzyme are enhanced by as much as two orders of magnitude upon differentiation of EC cells into endodermlike cells, particularly parietal endoderm (e.g., F9 cells treated with RA plus dbcAMP or micromolar levels of RA alone). Even when cells differentiate to other phenotypes there appears to be a transient (within 48 hr) but significant increase in plasminogen activator secretion. For these reasons this differentiation marker is useful for following retinoid-induced differentiation in early stages. Plasminogen activator is also a very sensitive parameter since elevated secretion of this enzyme by only a small percentage of cells in the culture can be readily detected. A quantitative assay has been used to measure secretion of plasminogen activator by EC cells. 16This assay measures plasminogen-dependent fibrinolysis of 125I-labeled fibrinogen. Cells are cultured and induced to t5 M. I. Sherman, S. Strickland, and E. Reich, Cancer Res. 36, 4208 (1976). 16 S. Strickland, E. Reich, and M. I. Sherman, Cell (Cambridge, Mass.) 9, 231 (1976).
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differentiate as described above. Twenty-four hours before a time point is to be taken, medium is removed, cells are rinsed thoroughly with PBS, and S-DMEM medium containing heat-inactivated, plasminogen-depleted serum 17 is added. After incubation, conditioned medium is collected and stored at - 2 0 ° and cells are counted. For the assay, a mixture of 1251labeled fibrinogen and unlabeled fibrinogen (30/zg, -4000 cpm//xg) in a 1 : 11 mixture of PBS/water is added to each well of 24-well plates and allowed to dry at 37° . Dried plates can be stored for several days at room temperature. Two hours prior to assay, 0.3 ml of a 10% solution of plasminogendepleted serum in 0.1 M Tris-HC1, pH 7.4, is added to each well. After a 2-hr incubation at 37°, each well is washed twice with 0.1 M Tris-HC1, pH 8.1. For measuring plasminogen activator secretion, 20/xl conditioned medium and 5 ttl plasminogen (10/zg) in 225 ~1 of 0.1 M Tris-HCl, pH 8.1, are added to each well and incubated for 2 hr at 37°. Reaction mixtures are collected, each well is washed with 250 /xl Tris buffer, and the wash solution is pooled with the reaction mixture. Samples are then counted in a 3' counter to determine the release of 1251. Samples should be run in triplicate. Two controls should always be included: in one, wells are incubated with 25/zl trypsin-EDTA solution to determine the total number of 1251counts per minute that can be released with protease; a second control consists of medium previously incubated without cells (also for 24 hr at 37°), to determine background levels of soluble 1251.When characterizing a new cell line, it is also prudent to include control samples lacking plasminogen to ensure that all fibrinolysis is plasmin-dependent. For quantitation, the solubilized counts in the control medium samples are subtracted from experimental values, and the results are normalized for trypsin-degradable 125I-labeled fibrin and expressed on a cell number basis. Plasminogen activator can also be qualitatively determined in situ by the fibrin-agar assay as outlined by Sherman et al. 15 Structural Proteins
There are several cell surface and cytoskeletal proteins for which antibodies are available. These proteins can be easily detected by indirect immunostaining. Fibronectin is synthesized by EC cells but is not retained in association with the cell surface. On cell differentiation into parietal endoderm, fibronectin can accumulate as part of the basement membrane.18 It also becomes surface-associated when fibroblastlike cells i7 L. Ossowski, J. C. Unkeless, A. Tobia, J. P. Quigley, D. B. Rifkin, and E. Reich, J. Exp. Med. 137, 112 (1973). 18 j. Wolfe, V. Mautner, B. Hogan, and R. Tilly, Exp. Cell Res. 118, 63 (1979).
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are formed. Increased synthesis and extracellular matrix deposition of three other basement membrane proteins, namely, collagen IV, laminin, and entactin, can be detected when EC cells differentiate into endodermlike cells) 9,2° On the other hand, a-fetoprotein antibodies are useful for staining visceral endodermlike cells. 21 These latter markers might be of more limited utility, depending on patterns of differentiation of the EC lines under study. Cytoskeletal proteins have also been employed to evaluate EC cell differentiation, although such markers are perhaps more useful as secondary indicators. Keratin intermediate filaments are detected by immunofluorescence when EC cells differentiate into epithelial cells. 22 Monoclonal antibodies TROMA-1 and TROMA-3 react with different intermediate filament components present in gut and fetal liver as well as mesoderm and endoderm-derived epithelia in adult animals. Both fail to react with EC cells, but they detect an ordered intermediate filament network present in differentiated cells: TROMA-1 reacts strongly with both visceral and parietal endoderm cells, whereas TROMA-3 is more specific, reacting only with parietal endoderm. 23 Two other intermediate filament components not expressed in EC cells, Endo A and Endo B, are readily detectable in trophoblast, visceral endoderm, parietal endoderm, and liver cells but not in myoblasts, neuroblastoma cells, or keratinocytes. 24 Differentiation of EC cells into glial-like cells and myoblastlike cells is accompanied by neurofilament and desmin expression, respectively. 25 mRNA Analyses cDNA probe technology can be utilized to evaluate EC cell differentiation. These probes can often be used to study differentiation at the molecular level before measurable morphological or biochemical changes occur. Several cDNA clones for genes either up- or downregulated have been reported, some representing structural proteins or enzymes previously known to be regulated, as described above (e.g., collagen IV, 19 A. Grover and E. D. Adamson, J. Biol. Chem. 260, 12252 (1985). 2o A. R. Cooper, A. Taylor, and B. L. M. Hogan, Dev. Biol. 99, 510 (1983). 2~ B. L. M. Hogan, A. Taylor, and E. Adamson, Nature (London) 291, 235 (1981). 22 D. Paalin, H. Jakob, F. Jacob, K. Weber, and M. Osborn, Differentiation 22, 90 (1982). 23 R. Kemler, P. Brulet, M. T. Schnebeley, J. Gaillard, and F. Jacob, J. Embryol. Exp. Morphol. 64, 45 (1981). 24 A. Grover, R. Oshima, and E. D. Adamson, ]. Cell Biol. 96, 1690 (1983). 25 E. M. V. Jones-Villeneuve, M. W. McBurney, K. A. Rogers, and V. 1. Kalnins, J. Cell Biol. 94, 253 (1982).
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laminin, entactin, a-fetoprotein), 26-28 others encoding novel proteins of u n k n o w n function. 27,29
For R N A isolation from EC cells, we have found a slightly modified version of the procedure described by Chomczynski and Sacchi 3° to be particularly useful and convenient. Because of its simplicity, it allows simultaneous processing of a large number of samples, as would be required, for example, in time-course experiments. Embryonal carcinoma cells are plated as described above (preferentially in culture dishes rather than flasks). At the end of the incubation period, cells are washed with PBS and lysed in situ by the addition of 2 ml/100-mm dish of guanidine thiocyanate solution (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). The lysate is then transferred with the help of a rubber policeman either to a siliconized and baked Corex 15-ml glass tube or to a disposable 14-ml polypropylene tube (Falcon 2059). The R N A is extracted by adding 0.2 ml of 2 M sodium acetate, pH 4.0, 2 ml water-saturated phenol, and 0.4 ml chloroform. After vortexing for 10 sec, the suspension is cooled on ice for 15 min and centrifuged at 8000 rpm (e.g., Sorval SS-34 rotor) for 20 rain at 4°. The aqueous phase containing the R N A is transferred to a new tube, mixed with 1 ml ice-cold 2propanol, and incubated at - 2 0 ° to precipitate the RNA. Care should be taken to leave behind the interphase, where DNA is present. The RNA is pelleted by centrifugation, resuspended in 0.3 ml guanidine thiocyanate solution, transferred to a 1.5-ml microcentrifuge tube and precipitated again with ! volume ice-cold 2-propanol or 2 volumes ice-cold ethanol. The RNA pellet is collected by centrifugation for l0 min in a microcentrifuge, washed once with 70% ice-cold ethanol, vacuum-dried, and dissolved in 50-100/xl water or 0.5% sodium dodecyl sulfate. If undissolved material is present at this point, the solution is centrifuged for 5 min in a microcentrifuge to pellet it, and the RNA can be further purified by ethanol precipitation from 0.3 M sodium acetate. Yields from a 100-mm dish vary from 100 to 200/xg of total RNA depending on cell density. The OD260/OD280 ratio in l0 m M Tris-HCl, pH 7.5, is normally not less than 2. RNA purified by this procedure is undegraded and stable for many months when stored at - 2 0 ° and can be used for cDNA synthesis, 26 S.-Y. Wang and L. Gudas, Proc. Natl. Acad. Sci. U.S.A. 80, 5880 (1983). 27 S.-Y. Wang, G. J. LaRosa, and L. J. Gudas, Dev. Biol. 107, 75 (1985). 28 p. R. Young and S. M. Tilghman, Mol. Cell. Biol. 4, 898 (1984). 29 R. A. Levine, G. J. La Rosa, and L. J. Gudas, Mol. Cell. Biol. 4, 2142 (1984). 30 p. Chomczynski and N. Sacchi, Anal. Biochern. 162, 156 (1987).
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Northern blots, poly(A) +, selection and S1 protection assays (see, e.g., Ref. 31). 31 j. F. Grippo and M. I. Sherman, this series, Vol. 190 [16].
[36] P a r a s i t e R e t i n o i d - B i n d i n g P r o t e i n s By BRAHMA P. SANI Introduction The major theory to explain the molecular mechanism of retinoid action in the control of epithelial differentiation and growth centers on the well-known cellular binding proteins for retinol and retinoic acid, CRBP and CRABP, respectively, 1-4 and on the recently discovered nuclear receptors for retinoic acid (RARs). 5-7 The role of retinoids in parasites is largely unknown although the occurrence and uptake of retinol and retinoic acid, as well as the formation of retinol from r-carotene by parasitic helminths, have been described by several investigators. Growth of some developmental stages of helminth parasites may depend on host levels of retinol. 8,9 Sturchler et al. 1o showed that the retinol concentration in adult O n c h o c e r c a v o l v u l u s was 8 times higher than that of the surrounding host tissues. Although the exact function of retinoids in parasites is not clear, it may be assumed, by analogy to host tissues, that they control certain vital biological functions of the parasites, such as differentiation, growth, and reproduction. In our efforts to study the interactions of retinoids with parasitic components, we discovered and partially characterized specific
I M. M. Bashor, D. O. Toft, and F. Chytil, Proc. Natl. Acad. Sci. U.S.A. 70, 3483 (1973). 2 B. P. Sani and D. L. Hill, Biochem. Biophys. Res. Commun. 61, 1276 (1974). 3 D. E. Ong and F. Chytil, J. Biol. Chem. 250, 6113 (1975). 4 B. P. Sani and D. L. Hill, Cancer Res. 36, 409 (1976). 5 M. Petkovich, N. J. Brand, A. Krust, and P. Chambon, Nature (London) 330, 444 (1987). 6 V. Giguere, E. S. Ong, P. Sequi, and R. M. Evans, Nature (London) 330, 624 (1987). 7 N. Brand, M. Petkovich, A. Krust, P. Chambon, H. Dethe, A. Marchio, P. Tiollais, and A. Dejean, Nature (London) 332, 850 (1988). 8 D. Mahalanabis, K. N. Jalan, T. K. Maitra, and S. R. Agarwal Am. J. Clin. Nutr. 29, 1372 (1976). 9 D. M. Storey, Z. Parasitenkd. 67, 309 (1982). 10 D. Sturchler, B. Holzer, A. Hanck, and A. Oegremont, Acta Trop. 40, 261 (1983).
METHODS IN ENZYMOLOGY, VOL. 189
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