IN VITROCELLULAR& DEVELOPMENTALBIOLOGY Volume 25, Number 10, October 1989 9 1989 Tissue Culture Association,Inc.
NUTRITIONAL REQUIREMENTS OF PAPILLOMAVIRUS-TRANSFORMED M O U S E C E L L S A N D AN U N I N F E C T E D P A R E N T L I N E IN SERUM-FREE CULTURE TERESA P. D{ LORENZO', JOSEPH A. DE MARO~, ANDDOROTHY E. PUMO3
Department of Biology, Hofstra University, Hempstead, New York 11550 {Received 19 September 1988; accepted 3 June 1989)
SUMMARY A serum-free culture system was used to compare the nutritional requirements of mouse mammary cells transformed by bovine papillomavirus type 1 (ID13 cells) and the uninfected parent line (C127 cells). The serum-free, chemically defined medium used for this study was an MCDB 151-based medium (MCDB 151+S-4-I), supplemented with epidermal growth factor, transferrin, hydrocortisone, ethanolamine, phosphoethanolamine, retinoic acid, trace metals, and insulin. Proliferation of either cell type in serum-free culture required the addition of 250 /ag/ml of insulin. ID13 cells have a doubling time of greater than 96 h in MCDB 1 5 1 + S + I , whereas C127 cells have a doubling time of 60 h. This is in sharp contrast to the growth characteristics of the two cell types in 10% fetal bovine serum, where doubling times for the ID13 and C127 cells are 24 and 30 h, respectively. Culture of the cells in a serum-free medium has therefore revealed that the papillomavirus-transformed cells have more stringent growth requirements than the uninfected parent line.
Key words: C127; transformation; papillomavirus; insulin; serum-free culture; neuropeptides. INTRODUCTION Papillomaviruses induce benign tumors, or papillomas, in many animal species including humans (20). In certain cases, papillomas may undergo malignant conversion to carcinomas (34,43). A cell culture system that allows the propagation of papillomaviruses does not yet exist. However, bovine papillomavirus type 1 (BPV-1) readily induces in vitro transformation of C127 cells, which are an established R I I I mouse line derived from breast tissue (11). Although the transformed cells are not productively infected by the virus, the viral DNA is maintained in the cells. Transformation by bovine papillomavirus type 1 in vitro is a model system which has been extensively studied to help understand the mechanism of transformation and the biologic activity of papillomaviruses during natural infection (2,8,9, 12,19,21,30,40,42). ID13 ceils are a line of C127 cells that have been transformed in vitro by BPV-1 (11). The C127 and ID13 cell lines comprise a model system that can be used to investigate the differences which exist between untransformed and BPV-transformed cells in vitro. The ID13 cells exhibit loss of density-dependent inhibition of growth, colony formation in soft agar, and tumor formation in
' Present address: Department of Otolaryngology, Long Island Jewish Medical Center, New Hyde Park, NY 11042. 2 Present address: Department of Neuropathology, Albert Einstein College of Medicine, Bronx, NY 10478. 3 To whom correspondence should be addressed.
nude mice (11). These characteristics are typical of cells that have undergone transformation in vitro (31). The C127 and ID13 cells are routinely grown in medium supplemented with 10% fetal bovine serum. Many transformed cells are capable of growth in low serum, presumably because they have a decreased requirement for hormones and growth factors (16,17). ID13 cells do not readily grow in low serum. Therefore we questioned whether the ID13 ceils have different requirements for serum factors than other transformed cells and whether the ID13 cells' nutritional requirements differ from those of the C127 cells. A serum-free, chemically defined medium to which the C127 and ID13 cells respond differently is described herein. The ID13 cells grow very poorly in a serum-free medium in which the C127 cells grow well, which suggests that the transformed (ID13) cells have more stringent requirements for growth than the C127 cells. MATERIALS AND METHODS
C127 and 11913 cells. C127 cells were obtained from the American Type Culture Collection, Rockville, M D " (CRL1616). ID13 cells are C127 cells transformed with BPV-1, which were obtained from Dr. P. Howley. The cells were routinely grown in medium containing Dulbecco's modified Eagle's (DME) medium (GIBCO, Grand Island, NY) with 4.5 mg of glucose/ml, supplemented with 10% fetal bovine serum (GIBCO) and an additional 2 m M glutamine (GIBCO). The cells were 909
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passaged every 3 to 4 d using trypsin-EDTA in Hanks' balanced salt solution without calcium or magnesium ~GIBCO). Media components. Experiments to develop a serumfree medium for the C127 and ID13 cells were performed in 24-well cell culture plates using MCDB 151 {Sigma, St. Louis, MO) ~98) as the basal medium. The following supplements were added initially: epidermal growth factor (Sigma~ 5 ng/ml, transferrin {Sigma) 100 /ag/ml, hydrocortisone lSigmaj 0.5 /ag/ml, ethanolamine (Sigma) 0.I mM, phosphoethanolamine {Sigma] 14 /ag/ml, insulin {Sigma) 5 /ag/ml, retinoic acid {a generous gift of Dr. Bettie Steinberg) 0.01 /aM, and calcium chloride {Sigma} 1.5 mM. The medium also contained the trace element mixture {stock L) from MCDB 104 ~24). Also added was 5 /al/ml of stock 1 (28~ containing essential amino acids. The final, complete medium was made fresh each day and is referred to as M C D B 151+S. MCDB 1 5 1 + S + I is identical to MCDB 151+S, except that the insulin concentration in MCDB 151-{-S+I is 250 /ag/ml {see Results). All components of the medium were prepared in distilled, deionized water, except for retinoic acid and hydroeortlsone which were solubilized in 100% ethanol, and epidermal growth factor which was dissolved in Hanks' balanced salt solution without calcium or magnesium. Stock solutions of individual medium supplements were aliquoted into smaller portions. Stock solutions were stored at --20 ~ C until needed, except stock L and the ealcinm chloride stock solution, which were stored at 4 ~ C. MCDB 105, FI2, gastrin, prolactin, and triiodothyronine were obtained from Sigma. Insulinlike growth factor II, substance P, and bombesin were obtained from Peninsula {Belmont, CA}. Transforming growth factor-/3 {TGF-/3) was obtained from Collaborative Research Lexington, MA). Growth assays. Confluent cultures were harvested on Day 1 of confiueney with trypsin-EDTA. The trypsin was inactivated with D M E containing 10% fetal bovine serum, and the cells were centrifuged at 200 X g at 4 ~ C for 5 rain. Cells were washed 3 times in phosphate buffered saline {PBS; 0.14 M sodium chloride, 0.01 M phosphate, pH 7.6} and counted using trypan blue exclusion ~0.25% wt/vol in PBS). Individual wells in 24-well plates were seeded with 400/al of cell suspensions adjusted to 5 X I04, 1 X I05, and 5 X l05 cells/ml in the medium to be tested. Wells were fed every 2 to 3 d with freshly prepared medium. The effects of various medium components on the growth of C127 and I D I 3 cells were determined by culturing the cells for 4 d. They were then washed with PBS, fixed with 4% formaldehyde in PBS for I0 rain, washed with PBS, and stained with 0.1% (wt/vol} crystal violet in water for 1 min. The plates were destained with cold water, and staining intensity was evaluated by comparing experimental wells with control wells within the same plate. If an additive or component seemed to stimulate the growth of C127 or ID13 cells {determined by an increase in staining intensity], cells were then plated in medium containing varying concentrations d the supplement. A
replicate plate of cells was counted every other day for 8 d using trypan blue exclusion to determine whether an increase in staining intensity correlated with a change in cell number. RESULTS Choice of basal medium. The growth response of the C127 and ID13 cells to a serum-free medium was first evaluated using MCDB 151+S {defined in Materials and Methods). However, growth comparable to that observed in the routine serum-containing medium did not occur in MCDB 151+S. To determine if a different basal medium would better stimulate cell growth, the basal medium in MCDB 151+S was replaced with other basal media. Stained plates of cells which had grown for 4 d were compared. Replacement of M C D B 151 with either MCDB 105 ioptimized for fibroblasts) 125~, DME I4.5 g glucose/liter} (10~, Ham's F12 I13}, or a one-to-one mixture of DME and F12 did not cause any observable change in stained surface area or in staining intensity over the 4-d period, regardless of the initial plating density. Likewise, replacement of MCDB 151 with mixtures of MCDB 105 and MCDB 151 in different ratios {3:1, 1:1, and 1:3) did not seem to enhance staining intensity. The basal medium used for the remainder of the experiments described was MCDB 151. MCDB 151 is a nutrient medium optimized to promote growth of epidermal keratinocytes (a type of epithelial cell) (28). The C127 cells have been shown to possess epithelial characteristics by immunocytochemistry and Western blot analysis of their intermediate filament proteins lunpublished data}. MCDB 151 was also chosen because the calcium concentration is low (30 ~aM), so the effects of low and high calcium concentrations on cell growth could be examined easily in this medium after appropriate supplementation with calcium chloride. Calcium concentration is known to affect the proliferation of a wide variety of cells in vitro, with epithelial cells especially sensitive to this ion ~5,15). When C127 and ID13 ceils were plated in MCDB 151+S with varying calcium concentrations (30/aM, 0.1 raM, 0.4 raM, or 1.5 mM), obvious effects on proliferation were not noted. However, plating efficiency increased as the calcium concentration increased, so 1.5 m M calcium was used in all subsequent experiments.
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Growth assays were next performed on C127 and ID13 cells in MCDB 151, supplemented with various concentrations of certain medium components. The concentration of one component at a time was varied; the concentrations of all other components were as stated for M C D B 151+S. The majority of supplements added to the MCDB 151 were chosen on the basis of the growth requirements of keratinocytes (5,37L Most of the additives were tested at 10 times less, and 10 and 100 times more than the amount present in MCDB 151+S. Also, most were individually omitted to determine if the component had an inhibitory effect on either C127 or ID13 cells. Growth assays were done using three concentrations each of epidermal growth factor {ranging from 2.5 to 500
SERUM-FREE C127 AND ID13 CULTURES
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FIG. I. Effect of insulin concentration on the growth of C127 and IDI3 cells. Cells were plated at 2 )< I04 cells/2-em 2 well in MCDB 15]-4-S, and the insulin concentration was varied as indicated. Control cells were plated in DME supplemented with 10% fetal bovine serum and 2 mM glutamine. Attached cells were counted on Day 8. Cell numbers shown are averages of six separate wells. Bars indicate I SD.
ng/ml), transferrin (1 to 1000 #g/ml), ethanolamine 40.01 to l0 mM), and phosphoethanolamine {l.41 to 1410 pg/ml). For both cell types, no change in staining intensity was observed at any of the concentrations tested when compared to the standard concentration present in M C D B 151-I-S. Increasing the glutamine concentration to 16 m M also did not lead to an increase in staining intensity at the end of 4 d. In contrast, high concentrations of insulin (250 to 500 /~g/ml) correlated with an increase in stained surface area after 4 d for both the C127 and the ID13 cells. In the absence of insulin, neither cell type was able to survive for the 4-d test period. Insulin amounts between 5 to 125 /~g/ml seemed to allow cell survival, but no quantitative differences in staining were apparent. Although an increase in stained surface area is often assumed to indicate cell proliferation, it might indicate an increase in cell size rather than cell number. Cell counts were made over an 8-d period to determine whether the increase in staining reflected an increase in cell number. Figure 1 shows that high concentrations of insulin {250 to 500 pg/ml) brought about an increase in cell number which differed significantly from that observed at 5 pg/ml. The difference between the cell number (growth yield) at Day 8 with 250 pg/ml vs. 500 pg/ml of insulin was insignificant (Student's t test, P > 0.1), so 250 pg/ml was used in all subsequent experiments. Insulin concentrations of 50 and 100 pg/ml were not significantly different than 5 /~g/ml with respect to stimulation of cell proliferation. M C D B 151-I-S with an insulin concentration of 250 pg/ml instead of 5 pg/ml will be referred to as M C D B 15I-FS-t-I. After 8 d, C127 cells grown in M C D B 15I-I-S-{-I grew to within one population doubling of the control cells grown in serum-containing medium. That is, M C D B 151+S-t-I supported growth of the C127 cells almost as well as did serum-containing medium. In contrast, ID13 cells showed fewer population doublings in M C D B 1 5 1 + S + I than did
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C127, and growth yield and growth rate were much lower than in the presence of serum. The difference in the growth curves of the C127 and ID13 cells in M C D B 1 5 1 + S + I and in serum-containing medium are shown in Fig. 2. Interestingly, the C127 cells consistently reached higher densities (approximately threefold higher) in M C D B 1 5 1 + S + I than the ID13 cells did. As shown in Fig. 2, this result is in marked contrast to the growth characteristics of the two cell types in the presence of serum. The other medium components tested above were retested for effects on proliferation of the C127 and I D I 3 cells using M C D B 151-4-S+I. The concentration of glucose in M C D B 151-4-S-4-I ( I . I mg/ml) is lower than in the serum-containing medium (4.0 mg/ml) in which the C127 and I D I 3 cells are routinely grown. However, increased glucose (4.5 to 21.5 mg/ml) did not improve serum-free growth of either cell line in MCDB 151-I-S-4-I. Therefore, we do not believe that glucose is rate-limiting for growth and is not the cause of the difference between growth in M C D B 1 5 1 + S + I and serum. The basal medium in M C D B 1 5 1 + S + I was also replaced by the basal media mentioned above (see "Choice of basal medium"). This did not improve serum-free growth of the C127 and ID13 cells. When the stock 1 amino acid mixture (28) was increased to 40 /~l/ml there was a minimal positive effect on the growth of both cell types. However, the effect was observed only at the lowest cell density. Varying
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FIG. 2. Growth curves of C127 (circles) and ID13 (triangles} cells in MCDB 151+S+I (open symbols) and in serumsupplemented DME (solid symbols). Cells were plated at 2 X 10' eells/2-cm 2 well. Each point represents an average of five or six separate wells. Bars indicate I SD.
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DILORENZOETAL.
concentrations of hydrocortisone and retinoic acid were also tested, but no increases in cell growth were detected. Effects of additional substances on growth. A number of other substances known to affect the proliferation of certain cell types I1,6,27,29,32,35,36) were also tested in an effort to improve serum-free growth of one or both of the two cell lines. These substances are not present in MCDB 1 5 1 + S + I . They include insulinlike growth factor II, substance P, bombesin, gastrin, prolactin, triiodothyronine, and transforming growth factor-/3. They were evaluated by adding to MCDB 1 5 1 + S + I at several concentrations. Neither the C127 nor ID13 cells showed a significant positive response to any of these factors. Therefore these factors are not the components of serum required by ID13 cells to achieve growth rates or saturation densities comparable to C127 cells.
DISCUSSION Progress in the study of papillomaviruses and papillomavirus infection has been slow because no cell culture system exists that allows the propagation of the viruses in the laboratory. C127 cells and their BPV-transformed counterparts, ID13 cells, comprise a model system that has been studied to help understand the mechanism of transformation and the activity of papillomaviruses during natural infection. We have characterized some of the nutritional requirements of these cells. Growth of either cell type required 250 pg/ml of insulin, in contrast to the optimal concentration of 5 ~g/ml reported for human and mouse epidermal keratinocytes {4,37), human nasal and bronchial epithelial cells (22,41), human ureter and bladder epithelial cells {18), and the 10 pg/ml reported for Chinese hamster ovary cells (26). Virtually every cell type tested to date requires insulin for growth in serum-free medium {3). In vitro, insulin regulates the transport of amino acids and glucose into the cell (7). Insulin has been shown to stimulate the transport of amino acids through the A system {33). It stimulates glucose transport by stimulating the translocation of the glucose carrier protein from the cytosol to the plasma membrane {38). It was possible that the high concentration of insulin required for serum-free growth of the C127 and ID13 cells might actually reflect a need for higher concentrations of glucose or amino acids in the medium. However, increased concentrations of glucose did not improve serum-free growth, and increased concentrations of the amino acids present in stock 1 had only a minimal effect. The concentration of insulin required for cell growth in nearly all culture systems is higher than the physiologic concentration, perhaps in part because insulin is quickly inactivated at 37~ C in cysteinecontaining medium {14). In addition, insulin may mimic insulinlike growth factors (IGFs), such as IGF-I (26). High insulin concentrations may be needed to occupy receptors that have a high affinity for an IGF but a lower affinity for insulin. The I G F - I I receptor does not bind insulin {23), but insulin stimulates the translocation of I G F - I I receptors from inside the cell to the cell surface
~39). I G F - I I did not significantly alter serum-free growth of the C127 and ID13 cells. This does not rule out the possibility that the high concentration of insulin required in MCDB 1 5 1 + S + I partially reflects a need for another insulinlike factor, such as IGF-I. The most significant differenee we saw in the nutritional requirements of the two cell types was in their ability to grow in the defined medium. C127 cells had a doubling time of about 60 h in MCDB 1 5 1 + S + I , compared to 30 h in 10o7o fetal bovine serum. ID13 cells had a doubling time of greater than 96 h in the defined medium and 24 h in 10O7o fetal bovine serum. Moreover, on Day 8 the growth yield of C127 was threefold higher than ID13 when grown in the defined medium. These were unexpected results, beeause among the classic characteristics of virally transformed cells in culture is growth to higher saturation densities and a decreased requirement for serum. In contrast, the ID13 cells, which were transformed in vitro by BPV-1, do not respond as well to MCDB 1 5 1 + S + I as the uninfeeted parent C127 cells do. Further experiments are needed to determine what is present in serum that is required by ID13 cells but not by C127 cells. Conversely, perhaps the C127 cells are more inhibited by a toxin present in serum than are ID13 cells. As deseribed in Results, we examined the effects of several growth faetors and other substances commonly needed by cells in serum-free culture, as well as other components of serum that are less often found to be required. However, the yield of ID13 cells in MCDB 1 5 1 + S + I was never comparable to that observed in serum. It is possible that a different clone of BPV-transformed cells would not show this poor growth response. However, it is also possible that the difference in nutritional requirements of the C127 and ID13 cells is specifically brought about by papillomavirus-induced transformation. Further study of this phenomenon may lead to new insights into the mechanism of in vitro transformation in general and papillomavirus-induced transformation in particular.
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The authors thank Dr. Bettie Steinberg and unidentified reviewers for critical reading of the manuscript. This work was supported in part by grant #l-P01 NS19214 from the National Institutes of Health, Bethesda, M D , N S F grant # R l l - 8 2 1 7 7 9 8 from the National Science Foundation, Washington, DC, and by a grant from the Otolaryngology Foundation.