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Chicken embryo extract mitigates growth and morphological changes in a spontaneously immortalized chicken embryo fibroblast cell line
MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Chicken Embryo Extract Mitigates Growth and Morphological Changes in a Spontaneously Immortalized Chicken Embryo Fibroblast Cell Line S. A. Christman, B.-W. Kong, M. M. Landry, D. N. Foster1 Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108 trations of CEE ≥ 100 g/mL inhibited growth of all cells tested. However, addition of 50 g of CEE/mL enhanced the growth rate and improved the morphology of the SC1 cells. Addition of CEE to the other immortal or primary CEF cells did not increase the growth rate or change their morphology. Analysis of mRNA expression revealed that SC-1 cells treated with 50 g of CEE/mL had lower levels of the p16INK4a alternate reading frame sequence (ARF) and E2F-1 than untreated SC-1 cells. The increased growth rate and improved morphology of the SC-1 cells achieved with CEE treatment were retained following removal of CEE, and these improvements should aid in increasing the utility of the SC-1 cell line as a cellular/molecular reagent.
(Key words: chicken embryo extract, immortal chicken embryo fibroblast cell line) 2005 Poultry Science 84:1423–1431
The availability of permanent, nontransformed cell lines lacking endogenous and exogenous viral genomes for studying the conversion to an immortal state and to evaluate the effects of viral infection has been limited. Traditionally, in the absence of a suitable avian cell line, primary chicken embryo fibroblasts (CEF) have been used for analysis. Like all normal diploid fibroblasts, primary CEF cells have a finite life span of ∼20 to 25 passages in culture. Although primary cells are normal, there are advantages to having a nontransformed cell line, which provides an unlimited supply of identical cells. A major disadvantage of using primary cells for vaccine production is that titers fluctuate from lot to lot. Unlike rodent cells, which spontaneously immortalize at a low but measurable frequency, spontaneous immortalization in avian cells is very rare, as it is in human cells (Campisi, 1999; Sherr and DePinho, 2000). To date, only 2 spontaneously immortalized CEF cell lines (DF1 and SC-1) exist (Himly et al., 1998; Schaefer-Klein et al., 1998; Kim et al., 2001a,b; S. A. Christman, B.-W.
Kong, M. M. Landry, and D. N. Foster, unpublished data). The genes involved in the spontaneous immortalization of these CEF cell lines have been studied (Kim et al., 2001a,b; S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data). Other nonvirally and nonchemically immortalized CEF cell lines (breast-derived CEF immortal, BCEFi; heart-derived CEF immortal, HCEFi) also have been established (Kim et al., 2001a). A major difference between the SC-1 cell line and the DF-1, HCEFi, and BCEFi cell lines is that the SC-1 cell line has a significantly lower rate of growth and less uniform morphology than the other cell lines. After 3 yr in culture, the growth rate of the SC-1 cells is significantly less than that of primary CEF, DF-1, HCEFi, or BCEFi cells. The morphology of DF-1, HCEFi, and BCEFi cells appears to be smaller and more compact than primary CEF or SC-1 cells. The morphology of SC1 cells is more similar to that of primary CEF cells, which display a more spindle-shaped morphology, yet they remain elongated. Although many other chicken embryo cell lines have been documented, the utility of these lines has been limited due to the method of transforma-
2005 Poultry Science Association, Inc. Received for publication February 17, 2005. Accepted for publication May 5, 2005. 1 To whom correspondence should be addressed: [email protected].
Abbreviation Key: ARF = p16INK4a alternate reading frame; BCEFi = breast-derived CEF immortal cell line; CEE = chicken embryo extract; CEF = chicken embryo fibroblast; HCEFi = heart-derived CEF immortal cell line; PDD = population doublings per day; RT = reverse transcription.
INTRODUCTION
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ABSTRACT The SC-1 spontaneously immortalized chicken embryo fibroblast (CEF) cell line has been established recently. Although this cell line had been in culture for over 3 yr, its growth rate has remained lower than that of primary CEF cells, and the morphology has not been as uniform as observed in primary cells. In the present study, the SC-1 cell line was treated with chicken embryo extract (CEE) to determine whether growth rates could be increased and cell morphology enhanced. The CEE also was tested on primary CEF cells, another spontaneously immortalized CEF cell line (DF-1), and on 2 other nonvirally and nonchemically immortalized CEF cell lines (BCEFi and HCEFi). Results indicated that concen-
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MATERIALS AND METHODS Culture Conditions and Cell Growth Analysis The primary and immortal CEF cells (SC-1, BCEFi, and HCEFi) were derived from a specific pathogen-free avian supply, whereas the immortal DF-1 CEF cells were derived from East Lansing Line 0 (ev-0) Leghorn layer embryos.2 All cells were grown in Dulbecco’s modified Eagle’s medium high glucose enriched with 10% fetal calf serum, 1% penicillin-streptomycin, and 2 mM Lglutamine. All cell culture reagents were purchased.3
2
Avian Disease and Oncology Laboratory, East Lansing, MI. 3 Invitrogen, Carlsbad, CA. 4 Intergen, Purchase, NY. 5 Stratagene, La Jolla, CA. 6 ImageJ 1.24O, 2001, NIH, Washington, DC.
The CEE was isolated from 6-d-old embryos under sterile conditions. The embryos were rinsed 3 times in PBS before homogenization through a 50-cc syringe. The homogenate was then passed through a 22-gauge needle several times. The homogenate was diluted in Dulbecco’s modified Eagle’s medium at a 1:1 (vol:vol) ratio and pelleted by centrifugation (6,000 × g) at 4°C for 15 min. The supernatant (CEE) was removed and saved. The cell pellet was then lysed to isolate additional growth factors. Briefly, the cell pellet was resuspended in 5 mL of deionized H2O and repelleted by centrifugation (6,000 × g) at 4°C for 15 min. Sodium chloride (5 M) was added to the supernatant at a 1:30 (vol:vol) ratio to restore physiological salt concentration and was combined with the original CEE. The CEE was passed through a 0.45m filter for sterilization, and the protein concentration was determined using the Bradford method. Chicken embryo extract was frozen at −20°C and thawed prior to use. To determine growth rates, SC-1 CEF cells were plated at 3 × 105 cells/10-cm dish using culture conditions as described above. At 80% confluency, cells were trypsinized and counted, and the number of population doublings per day was calculated. The CEE was first added to SC-1 cells at passage 45. At passage 80, CEE was withdrawn from a subset of cells.
Morphological Analysis Images of live cells were captured using phase-contrast microscopy (100×). Morphological analysis of the cells was accomplished by comparing cell size.
RT-PCR Analysis For semiquantitative RT-PCR, 3 g of RNA treated with DNase I was converted to cDNA using Superscript II RT3 by following the manufacturer’s instructions. A portion (1 L) of the RT reaction was used to amplify cDNA fragments with chicken-specific primers (Table 1). All semiquantitative cDNA fragments were amplified using TaKaRa Ex Taq.4 The PCR products were verified to be in the linear range and were visualized by ethidium bromide staining. To validate the above quantitation method, PCR products also were amplified for 15 cycles (undetectable by ethidium bromide staining) and were detected by standard Southern blot hybridization using their corresponding [α-32P]-labeled cDNA probes prepared by RT-PCR (data not shown). Images were processed using the Eagle Eye II still video system.5 A minimum of 3 independent RT-PCR experiments were analyzed using the NIH image software program6 and normalized using the expression levels of gylceraldehyde-3-phosphate dehydrogenase (GAPDH).
Statistical Analyses Serial data generated from cells were analyzed statistically to determine treatment effects using Proc GLM in a two-way ANOVA for repeated measures (SAS/
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tion. Most have been immortalized via viral or chemical methods (Akiyama et al., 1973; Nazerian and Witter, 1975; Dinowitz, 1977; Nazerian et al., 1977; Kaaden et al., 1982; Gionti et al., 1989; Barald et al., 1997; Moscovici et al., 1977), providing a significant obstacle for molecular analysis. The recently developed spontaneously immortalized SC-1 cell line is free of endogenous viruses and is not transformed. However, the decreased growth rate has been a concern in terms of the line’s utility. The present study describes an attempt to increase the growth rate and improve the morphological characteristics of the spontaneously immortalized SC-1 CEF cell line. A critical requirement for the growth of cells in culture is the presence of appropriate growth factors. In the case of mammalian hemopoietic cells, these activities have been extensively investigated (see Clark and Kamen, 1987), and many growth factors have been identified, sequenced, and produced recombinantly. Chicken embryo extract (CEE) contains many growth factors that stimulate cell growth. This is evidenced by the fact that after fertilization, a chicken develops in only 21 d with all necessary growth factors provided in the egg. It was hypothesized that addition of CEE to standard growth medium might enhance the growth rate of the SC-1 cell line. The growth rates of mouse neural crest cells and human muscle cells have been shown to increase when CEE is present in the culture medium (Maxwell et al., 1996; Yasin et al., 1981). The number of growth factors identified in CEE increases constantly, and the combinatorial needs of cells grown in culture are being investigated. In the present study, CEE was isolated and used to determine whether the growth rate and morphology of the spontaneously immortalized SC-1 cell line could be enhanced. Analysis of genes involved in the p53 and Rb pathways was performed using reverse transcription (RT)-PCR to elucidate the mechanisms by which CEE may alter the expression of important cell cycle regulatory genes.
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Accession number
Primers1
Annealing temperature
NM_205264
F5′-CCGTGGCCGTCTATAAGAAA-3′ R5′-GCAGGAACTGTTGCACATGA-3′
60°C
MDM2
AF005045
F5′-CAATCAACAAGACTCTACGCTGGCTG-3′ R5′-TCATCTTCATCTGTGAGCTCCTGTCC-3′
65°C
p21
NM_204396
F5′-ACTTCAACTTCGAGACCGAGAC-3′ R5′-GGGCTCTTCCTATACATTGCAC-3′
58°C
p27
NM_204256
F5′-CATGCCAGAGGAAGTGGAAT-3′ R5′-TTCGGCCTACACAGTGAGTG-3′
55°C
Rb
AF323706
F5′-GGAGTCCAAGTTCCAACAAAAG-3′ R5′-ACAAACAAACACAGTGTCCCAG-3′
60°C
p15INK4b
NM_204433
F5′-GGGATTAGAGGGATGTGTGG-3′ R5′-GGCAGGAGGTGTTTACCAAA-3′
60°C
E2F-1
NM_205219
F5′-CCTCACCTCACCCATCCCTACCC-3′ R5′-GTGACAGTGCAATGAACTCATCCGC-3′
60°C
ARF
NM_204434
F5′-CCCGTAGAGCTTTGCAGTTC-3′ R5′-ACATGCAGGAACGGATCTTC-3′
60°C
1
F = forward; R = reverse.
STAT7). Differences in mRNA expression for each gene were determined using Proc GLM in a one-way ANOVA. Probabilities ≤ 0.05 were considered significant.
RESULTS Growth Rate and Morphological Analysis The morphology of the SC-1 cells was compared with primary CEF cells, immortal HCEFi and BCEFi cells, and the DF-1 cell line (the only other RT-negative spontaneously immortalized chicken cell line to have been established). The immortal DF-1 (passage 262), BCEFi (passage 120), and HCEFi (passage 96) had similar morphologies. These immortal cells appeared to be smaller and more uniform than primary passage 6 CEF cells, which displayed a spindle-shaped morphology, which are phenotypical of fibroblasts (Figure 1a). Immortal SC1 cells (passage 125) were elongated and possessed a high cytoplasmic:nuclear volume ratio, indicative of early senescent cells (Figure 1a). Treatment of the primary CEF cells and immortal DF1, HCEFi, and BCEFi cells with varying concentrations of CEE resulted in no phenotypical differences. Similarly, treatment of the SC-1 cells with 10 or 25 g of CEE/mL resulted in no phenotypical differences compared with untreated SC-1 cells. However, SC-1 cells treated with 50 g of CEE/mL were smaller, had a lower cytoplasmic:nuclear volume ratio than untreated cells, and were more spindle-shaped, characteristic of fibroblasts. The CEE-treated SC-1 cells at 50 g/mL also tended to display more contact (arrow in Figure 1a) with each other compared with untreated SC-1 cells.
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SAS/STAT, 1990, Version 9.1, SAS Institute Inc., Cary, NC.
The average growth rate of the primary CEF cells was 1.00 ± 0.13 population doublings per day (PDD), whereas the average growth rate of the SC-1 cells from passage 120 to 122 was only 0.60 ± 0.07 PDD (P < 0.05). The growth rates of the other immortal cells also were greater than that of the SC-1 cells with the DF-1 cells averaging 1.38 ± 0.10 PDD from passages 262 to 274, the HCEFi averaging 1.00 ± 0.09 PDD from passages 96 to 108, and the BCEFi averaging 1.22 ± 0.10 PDD from passages 120 to 132 (P < 0.05; Figure 1b). After 3 yr in culture, the growth rate of the SC-1 cells had increased from 0.20 PDD at passage 40 to 0.60 PDD at passage 107, which has been maintained to the current passage 160. Various concentrations of CEE were tested to determine which, if any, would increase growth rate. The concentrations of CEE tested ranged from 0 to 500 g/mL. In primary cells, concentrations of CEE > 100 g/mL caused cells to senesce earlier than untreated cells (P < 0.05; Figure 2a). In all immortal cells, concentrations of CEE ≥ 100 g/mL inhibited growth (data not shown). Addition of CEE to DF-1, BCEFi, and HCEFi cells at < 100 g/mL did not alter the growth rate of the cells. Chicken embryo extract was first added to the SC-1 cells at passage 45. By using a repeated measures test, addition of 10 or 25 g of CEE/mL appeared to have no significant effect on the growth rate of the SC1 cells, even after 80 passages in culture (P > 0.05; Figure 3a). However, addition of 50 g of CEE/mL significantly enhanced the growth rate of the SC-1 cells after 80 passages in culture (P < 0.05; Figure 3b). The average growth rate increased from 0.60 ± 0.07 PDD without CEE to 0.78 ± 0.06 PDD with 50 g of CEE/mL (P < 0.05). After pretreatment of the SC-1 cells with 50 g of CEE/mL for 40 passages, the CEE was withdrawn. After withdrawal of CEE for 40 passages, the growth rate of SC1 cells remained elevated compared with untreated SC-
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p53
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FIGURE 1. (a) Representative photomicrographs of primary passage 6 chicken embryo fibroblast (CEF) cells and immortal passage 262 DF-1 cells, passage 125 SC-1 cells, and passage 125 SC-1 cells + 50 g of chicken embryo extract (CEE)/mL. Bars represent 100 m. The arrow indicates increased cell contact. (b) Average growth rate of primary CEF (passages 4 to 12) and immortal SC-1 (passages 120 to 132), DF-1 (passages 262 to 274), breast-derived CEF immortal line (BCEFi; passages 120 to 132), and heart-derived CEF immortal cell line (HCEFi; passages 96 to 108) cells. Bars indicate SD. a-cBars with different letters indicate significant differences.
1 cells (Figure 3c). The growth enhancement that was observed in the SC-1 cells that had the CEE withdrawn continues to date.
mRNA Expression Analysis Because high levels of CEE (≥100 g/mL) caused early senescence in primary CEF cells, mRNA expression lev-
els of various cell cycle regulatory genes were measured. In primary CEF cells, levels of p53 (68 ± 4%), p16INK4a alternate reading frame sequence (ARF; 67 ± 3%), p15INK4b (87 ± 4%), Rb (60 ± 3%), and E2F-1 (76 ± 3%) mRNA expression were increased (P < 0.05) with addition of greater concentrations of CEE (100 g/mL), whereas expression of MDM2, p21, and p27 mRNA were decreased 30 ± 5% (P < 0.05; Figure 2b).
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duction in the expression of E2F-1 mRNA that was maintained after withdrawal of the CEE for 40 passages (Figure 4).
DISCUSSION
In the SC-1 cells, levels of p53, MDM2, p21, and p27 mRNA were decreased compared with primary CEF cells (S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data) and did not change significantly with varying concentrations of CEE or upon removal of CEE (Figure 4). The levels of ARF mRNA in the SC-1 cells were twice that measured in primary CEF cells (S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data). Increasing amounts of CEE decreased the expression of ARF mRNA in the SC-1 cells with 50 g of CEE/mL resulting in a 3-fold decrease in ARF expression compared with untreated SC-1 cells. The reduction in ARF mRNA expression in the presence of 50 g of CEE/mL was maintained even after withdrawal of the CEE for 40 passages. In SC-1 cells, the levels of Rb and p15INK4b were unchanged with varying concentrations of CEE (data not shown), whereas increasing concentrations of CEE caused a re-
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FIGURE 2. (a) Growth curve of primary chicken embryo fibroblast (CEF) cells with and without 100 g of chicken embryo extract (CEE)/ mL. The cells were thawed at passage 2 and then plated at a density of 3 × 105 cells/10-cm dish. At 80% confluency cells were trypsinized and counted, and the number of population doublings per day was calculated. (b) Relative mRNA expression levels of cell cycle regulatory genes for passage 6 CEF cells (− and + 100 g of CEE/mL). For each gene analyzed, the expression level was set to 1.00 for passage 6 CEF cells that had not been treated with CEE. The levels of the sample that had been treated with 100 g of CEE/mL were adjusted accordingly. A minimum of 3 independent reverse transcription-PCR experiments were analyzed using the NIH image software program (ImageJ 1.24O, 2001, NIH, Washington, DC) and normalized using the expression levels of gylceraldehyde-3-phosphate dehydrogenase. Differences ≤ 0.05 were considered significant.a,bBars with different letters indicate significant differences.
In the present study the spontaneously immortalized SC-1 cell line was treated with CEE in an attempt in increase growth rate and improve morphology. Because the availability of nonvirally and nonchemically derived cell lines is limited, creation of such cell lines is invaluable to provide suitable substrates for cellular and molecular analysis and for virus propagation and vaccine production. In fact, a pharmaceutical company has propagated Marek’s virus in the SC-1 cells and shown that titers were similar to those in primary CEF and immortal DF-1 cells (data not shown). The SC-1 cell line is only the second RT-negative spontaneously immortalized CEF cell line that has been reported. However, its slow growth rate has been a limitation to its utility as a cellular or molecular reagent. In an attempt to increase the growth rate of the SC-1 cell line to a rate that was more similar to primary CEF cells or other immortal cells (DF1, HCEFi, and BCEFi), the SC-1 cell line was treated with CEE. It was discovered that treatment of any of the cells with ≥100 g of CEE/mL restricted growth, suggesting that high concentrations of factors present in CEE are inhibitory to the growth of primary and immortal CEF cells. The primary CEF cells and immortal DF-1, HCEFi, and BCEFi cells did not benefit from the addition of CEE to the basal medium, indicating that these cells did not require additional growth factors present in CEE for optimum growth. On the other hand, the slower growth rate of the SC-1 cells suggested that the lack 1 or more growth factors was constraining the growth rate of the cells. To this end, the SC-1 cells were treated with CEE in an attempt to attain a growth rate that was more similar to the growth rate of primary CEF and other immortal CEF cells. In fact, the growth rate of the SC1 cells was enhanced by treatment with 50 g of CEE/ mL. It appeared that this concentration of CEE provided the appropriate concentration of growth factors that were beneficial for the growth of SC-1 cells and were deficient in basal media. The population of SC-1 cells was not as uniform as the primary CEF cells or the immortal DF-1, HCEFi, or BCEFi cells. The DF-1, BCEFi, and HCEFi cell lines had a smaller and more compact morphology than primary CEF or SC-1 cells. The SC-1 cells were more similar to primary CEF cells than were any of the other immortal cells examined. However, the SC-1 cell line was composed of some flattened and enlarged cells that had an increased cytoplasmic:nuclear volume ratio. Treatment of the SC-1 cells with 50 g of CEE/mL reduced the number of these flattened and enlarged cells, suggesting that this concentration of CEE was most beneficial in improving the morphology of the SC-1 cell line. Treatment of the primary CEF cells and immortal DF-1,
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FIGURE 3. (a) Growth analysis of SC-1 cells (passages 125 to 142) with varying concentrations of chicken embryo extract (CEE). (b) Growth analysis of the SC-1 cells (passages 125 to 142) with and without 50 g of CEE/mL. (c). Growth analysis of SC-1 cells (passages 125 to 142) + 50 g of CEE/mL and SC-1 cells that had been pretreated with 50 g of CEE/mL for 40 passages and then had the CEE withdrawn for 40 passages. Serial data generated from cells were analyzed statistically to determine treatment effects using Proc GLM in a 2-way ANOVA for repeated measures (SAS/STAT, 1990, Version 9.1, SAS Institute Inc., Cary, NC). Differences ≤ 0.05 were considered significant.
EMBRYO EXTRACT ENHANCES GROWTH AND MORPHOLOGY
BCEFi, and HCEFi cells with varying concentrations of CEE did not alter the morphology of these cells. Again, this finding suggested that these cells did not require additional growth factors deficient in basal media. An unexpected observation was that treatment of primary CEF cells with ≥100 g of CEE/mL prompted early onset of senescence. To gain a better understanding of this phenomenon in primary CEF cells, the mRNA expression levels of various cell cycle regulatory genes were examined via RT-PCR. The tumor suppressor p53 initiates pathways that result in cell senescence or death (Sherr and DePinho, 2000). As expected, levels of p53 mRNA were increased in primary CEF cells treated with ≥100 g of CEE/mL. Another important regulatory gene in the p53 pathway is MDM2, a proto-oncogene that is elevated in 30 to 40% of human tumors (Oliner et al., 1992). The MDM2 gene product binds to p53, inactivating its transcriptional activity (Momand et al., 1992; Oliner et al., 1993). Therefore, it would be expected that MDM2 should be decreased in senescent cells. As predicted, levels of MDM2 were decreased in primary CEF cells that were treated with concentrations of CEE ≥ 100 g/mL, again characteristic of senescent cells. The p21 (El-Diery et al., 1993; Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993a) and p27 (Polyak et al., 1994; Slingerland et al., 1994) genes belong to the Kip/ Cip family of CDK-inhibitory proteins that block transition from the G1 to the S phase of the cell cycle. In normal cells, DNA damage causes cell cycle arrest at the G1/S phase of the cell cycle via p53 stimulation of p21 expression (El-Deiry et al., 1993, 1994). It has been shown that levels of p21 and p27 are highest at senescence (Noda et al., 1994) and that over-expression induces cell cycle arrest at the G1/S phase of the cell cycle (El-Deiry et al., 1993, 1994; Polyak et al., 1994; Toyoshima and Hunter, 1994). However, in the present study, the levels of p21 and p27 were decreased in primary CEF cells that had been treated with ≥100 g of CEE/mL. It is possible that elevated levels of p53 initiated other pathways to trigger early senescence in the CEF cells that were treated with ≥100 g of CEE/mL. The ARF gene product functions upstream of MDM2 and p53 by stably binding to MDM2, thus preventing MDM2 from exporting p53 to the cytoplasm for degradation (Tao and Levine, 1999; Weber et al., 1999; Zhang and Xiong, 1999). In fact, it has been reported that ARF is required for senescence and is inactivated in many human tumors (Kamijo et al., 1997). Here, as expected, it was observed that levels of ARF mRNA were increased in primary CEF cells treated with ≥100 g of CEE/mL that entered senescence early. Another important gene that is involved in senescence is p16INK4a. Because chickens encode p15INK4b in place of p16INK4a (Kim et al., 2003), levels of p15INK4b were measured. As expected the levels of p15INK4b were elevated in primary CEF cells treated with ≥100 g of CEE/ mL that entered senescence early. It is likely that the increased expression of p53, ARF, and p15INK4b mRNA
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FIGURE 4. Relative mRNA expression levels of cell cycle regulatory genes for passage 125 SC-1 cells. The amount of chicken embryo extract (CEE; g/mL) is shown at the bottom of the figure (w50 = cells treated with 50 g of CEE/mL for 40 passages and then had the CEE withdrawn for 40 passages). For each gene analyzed, the expression level was set to 1.00 for SC-1 cells that had not been treated with CEE. The levels for other samples were adjusted accordingly. A minimum of 3 independent reverse transcription-PCR experiments were analyzed using the NIH image software program (ImageJ, 1.24O, 2001, NIH, Washington, DC) and normalized using the expression levels of gylceraldehyde-3-phosphate dehydrogenase. Differences ≤ 0.05 were considered significant. a–c Bars with different letters indicate significant differences.
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Clark, C., and R. Kamen. 1987. The human hematopoietic colony-stimulating factors. Science 236:1229–1237. Dinowitz, M. 1977. A continuous line of Rous sarcoma virustransformed chick embryo cells. J. Natl. Cancer Inst. 58:307–312. El-Deiry, W. S., J. W. Harper, P. M. O’Connor, V. E. Velculescu, C. E. Canman, et al. 1994. WAF1/CIP1 is induced in p53mediated G1 arrest and apoptosis. Cancer Res. 54:1169–1174 El-Deiry, W. S., T. Tokino, V. Velculescu, D. Levy, R. Parsons, et al. 1993. WAF1, a potential mediator of p53 tumor suppression. Cell 75:817–825. Gionti, E., P. Jullien, G. Pontarelli, and M. Sanchez. 1989. A continuous line of chicken embryo cells derived from a chondrocyte culture infected with RSV. Cell Differ. Dev. 27:215–223. Gu, Y., C. Turek, and D. Morgan. 1993. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 366:707–710. Harper, J. W., G. Adami, N. Wei, K. Keyomarse, and S. Elledge. 1993. The p21 cdk- interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816. Himly, M., D. N. Foster, I. Bottoli, J. S. Lacovoni, and P. K. Vogt. 1998. The DF-1 chicken fibroblast cell line: Transformation induced by diverse oncogenes and cell death resulting from infection by avian leucosis viruses. Virology 248:295–304. Kaaden, O. R., S. Lange, and B. Stiburek. 1982. Establishment and characterization of chicken embryo fibroblast clone LSCC-H32. In Vitro 18:827–834. Kamijo, T., F. Zindy, M. F. Roussel, D. E. Quelle, J. R. Downing, R. A. Ashmun, G. Grosveld, and C. J. Sherr. 1997. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91:649–659. Kim, H., S. You, I.-J. Kim, J. Farris, L. K. Foster, and D. N. Foster. 2001a. Increased mitochondrial encoded gene transcription in immortal DF-1 cells. Exp. Cell Res. 265:339–347. Kim, H., S. You, I.-J. Kim, L. K. Foster, J. Farris, S. Ambady, F. A. Ponce de Leon, and D. N. Foster. 2001b. Alterations in p53 and E2F-1 function common to immortalized chicken embryo fibroblasts. Oncogene 20:2671–2682. Kim, S.-H., M. Mitchell, H. Fujii, S. Llanos, and G. Peters. 2003. Absence of p16INK4a and truncation of ARF tumor suppressors in chickens. Proc. Natl. Acad. Sci. USA 100:211–216. Maxwell, G. D., K. Reid, A. Elefanty, P. F. Bartlett, and M. Murphy. 1996. Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures. Proc. Natl. Acad. Sci. USA 93:13274–13279. Momand, J., G. P. Zambetti, D. C. Olson, D. George, and A. J. Levine. 1992. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell 69:1237–1245. Moscovici, C., M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt. 1977. Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail. Cell 11:95–103. Nazerian, K., E. A. Stephens, J. M. Sharma, L. F. Lee, M. Gailitis, and R. L. Witter. 1977. A nonproducer T lymphoblastoid cell line from Marek’s disease transplantable tumor (JMV). Avian Dis. 21:69–76. Nazerian, K., and R. L. Witter. 1975. Properties of a chicken lympho-blastoid cell line from Marek’s disease tumor. J. Natl. Cancer Inst. 54:453–458. Noda, A., Y. Ning, S. Venable, O. Pereira-Smith, and J. Smith. 1994. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res. 211:90–98. Oliner, J. D., K. W. Kinzler, P. S. Meltzer, D. L. George, and B. Vogelstein. 1992. Amplification of a gene encoding a p53associated protein in human sarcomas. Nature 358:80–83.
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contributed to early onset of senescence in the primary CEF cells treated with ≥100 g of CEE/mL. In the SC-1 cells, varying concentrations of CEE (0, 10, 25, and 50 g/mL) did not significantly alter the expression of p53, MDM2, p21, p27, or p15INK4b. This was not surprising because expression of most of these genes were already lower than the expression in primary CEF cells (S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data). However, increasing concentrations of CEE lowered the expression of ARF and E2F-1. The expression of ARF in untreated SC1 cells was increased compared with primary CEF cells, whereas most immortal cells display reduced levels of ARF mRNA (Kamijo et al., 1997). Treatment of the SC1 cells with CEE decreased the expression of ARF mRNA to correspond to levels observed in other immortal cells (Kamijo et al., 1997). The lower levels of E2F-1 that were observed as the concentration of CEE increased were not expected. Previous work in our laboratory has shown that mRNA, protein, and functional activity of E2F-1 were dramatically elevated in all immortal cells studied (Kim et al., 2001b). However, a link between E2F-1 and ARF expression may explain the lower levels of E2F-1 observed in the SC-1 cells as the levels of ARF decreased (Bates et al., 1998). Another important finding of this study was that the increased growth rate, improved morphology, and pattern of gene expression were maintained in the SC-1 cells that were treated with 50 g/mL for 40 passages and then had the CEE withdrawn for 40 passages. This result suggested that treatment of the SC-1 cells with the appropriate concentration of CEE (50 g/mL) provided the cells with growth factors that altered gene expression to allow the SC-1 cells to improve their growth rate and enhance their morphology and that this improvement was imprinted and retained upon CEE withdrawal. In conclusion, it appears that treatment of the slowergrowing SC-1 cell line with CEE was successful in increasing the growth rate and improving the morphology of the cells and that the improvements achieved through CEE treatment were retained even after CEE withdrawal. This progression of the SC-1 cell line should aid in its utility as an appropriate model for investigations into cell cycle regulation and virus propagation.
EMBRYO EXTRACT ENHANCES GROWTH AND MORPHOLOGY Oliner, J. D., J. A. Pietenpol, S. Thiagalingam, J. Gyuris, K. W. Kinzler, and B. Vogelstein. 1993. Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature 362:857–860. Polyak, K., M.-H. Lee, H. Erdjument-Bromage, A. Koff, J. M. Roberts, P. Tempst, and J. Massague´. 1994. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78:59–66. Schaefer-Klein, J., I. Givol, E. V. Barsov, J. M. Whitcomb, M. VanBrocklin, D. N. Foster, M. J. Federspiel, and S. H. Hughes. 1998. The EV-O-derived cell line DF-1 supports the efficient replication of avian leucosis-sarcoma viruses and vectors. Virology 248:305–311. Sherr, C. J., and R. A. DePinho. 2000. Cellular senescence: Mitotic clock or culture shock? Cell 102:407–410. Slingerland, J., L. Hengst, C. Pan, D. Alexander, M. Stampfer, and S. Reed. 1994. A novel inhibitor of cyclin-cdk activity
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deteced in transforming growth factor β- arrested epithelial cells. Mol. Cell. Biol. 14:3683–3694. Tao, W., and A. J. Levine. 1999. p19ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc. Natl. Acad. Sci. USA 96:6937–6941. Toyoshima, H., and T. Hunter. 1994. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78:67–74. Weber, J., L. Taylor, M. Roussel, C. Sherr, and D. Bar-Sagi. 1999. Nucleolar Arf sequesters Mdm2 and activates p53. Nat. Cell Biol. 1:20–26. Xiong, Y., H. Zhang, and D. Beach. 1993a. Subunit rearrangement of the cyclin- dependent kinases is associated with cellular transformation. Genes Dev. 7:1572–1583. Yasin, R., D. Kundu, and E. J. Thompson. 1981. Growth of adult human cells in culture at clonal densities. Cell Differ. 10:131–137. Zhang, Y., and Y. Xiong. 1999. Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol. Cell 3:579–591.
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(Key words: chicken embryo extract, immortal chicken embryo fibroblast cell line) 2005 Poultry Science 84:1423–1431
The availability of permanent, nontransformed cell lines lacking endogenous and exogenous viral genomes for studying the conversion to an immortal state and to evaluate the effects of viral infection has been limited. Traditionally, in the absence of a suitable avian cell line, primary chicken embryo fibroblasts (CEF) have been used for analysis. Like all normal diploid fibroblasts, primary CEF cells have a finite life span of ∼20 to 25 passages in culture. Although primary cells are normal, there are advantages to having a nontransformed cell line, which provides an unlimited supply of identical cells. A major disadvantage of using primary cells for vaccine production is that titers fluctuate from lot to lot. Unlike rodent cells, which spontaneously immortalize at a low but measurable frequency, spontaneous immortalization in avian cells is very rare, as it is in human cells (Campisi, 1999; Sherr and DePinho, 2000). To date, only 2 spontaneously immortalized CEF cell lines (DF1 and SC-1) exist (Himly et al., 1998; Schaefer-Klein et al., 1998; Kim et al., 2001a,b; S. A. Christman, B.-W.
Kong, M. M. Landry, and D. N. Foster, unpublished data). The genes involved in the spontaneous immortalization of these CEF cell lines have been studied (Kim et al., 2001a,b; S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data). Other nonvirally and nonchemically immortalized CEF cell lines (breast-derived CEF immortal, BCEFi; heart-derived CEF immortal, HCEFi) also have been established (Kim et al., 2001a). A major difference between the SC-1 cell line and the DF-1, HCEFi, and BCEFi cell lines is that the SC-1 cell line has a significantly lower rate of growth and less uniform morphology than the other cell lines. After 3 yr in culture, the growth rate of the SC-1 cells is significantly less than that of primary CEF, DF-1, HCEFi, or BCEFi cells. The morphology of DF-1, HCEFi, and BCEFi cells appears to be smaller and more compact than primary CEF or SC-1 cells. The morphology of SC1 cells is more similar to that of primary CEF cells, which display a more spindle-shaped morphology, yet they remain elongated. Although many other chicken embryo cell lines have been documented, the utility of these lines has been limited due to the method of transforma-
2005 Poultry Science Association, Inc. Received for publication February 17, 2005. Accepted for publication May 5, 2005. 1 To whom correspondence should be addressed: [email protected].
Abbreviation Key: ARF = p16INK4a alternate reading frame; BCEFi = breast-derived CEF immortal cell line; CEE = chicken embryo extract; CEF = chicken embryo fibroblast; HCEFi = heart-derived CEF immortal cell line; PDD = population doublings per day; RT = reverse transcription.
INTRODUCTION
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ABSTRACT The SC-1 spontaneously immortalized chicken embryo fibroblast (CEF) cell line has been established recently. Although this cell line had been in culture for over 3 yr, its growth rate has remained lower than that of primary CEF cells, and the morphology has not been as uniform as observed in primary cells. In the present study, the SC-1 cell line was treated with chicken embryo extract (CEE) to determine whether growth rates could be increased and cell morphology enhanced. The CEE also was tested on primary CEF cells, another spontaneously immortalized CEF cell line (DF-1), and on 2 other nonvirally and nonchemically immortalized CEF cell lines (BCEFi and HCEFi). Results indicated that concen-
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MATERIALS AND METHODS Culture Conditions and Cell Growth Analysis The primary and immortal CEF cells (SC-1, BCEFi, and HCEFi) were derived from a specific pathogen-free avian supply, whereas the immortal DF-1 CEF cells were derived from East Lansing Line 0 (ev-0) Leghorn layer embryos.2 All cells were grown in Dulbecco’s modified Eagle’s medium high glucose enriched with 10% fetal calf serum, 1% penicillin-streptomycin, and 2 mM Lglutamine. All cell culture reagents were purchased.3
2
Avian Disease and Oncology Laboratory, East Lansing, MI. 3 Invitrogen, Carlsbad, CA. 4 Intergen, Purchase, NY. 5 Stratagene, La Jolla, CA. 6 ImageJ 1.24O, 2001, NIH, Washington, DC.
The CEE was isolated from 6-d-old embryos under sterile conditions. The embryos were rinsed 3 times in PBS before homogenization through a 50-cc syringe. The homogenate was then passed through a 22-gauge needle several times. The homogenate was diluted in Dulbecco’s modified Eagle’s medium at a 1:1 (vol:vol) ratio and pelleted by centrifugation (6,000 × g) at 4°C for 15 min. The supernatant (CEE) was removed and saved. The cell pellet was then lysed to isolate additional growth factors. Briefly, the cell pellet was resuspended in 5 mL of deionized H2O and repelleted by centrifugation (6,000 × g) at 4°C for 15 min. Sodium chloride (5 M) was added to the supernatant at a 1:30 (vol:vol) ratio to restore physiological salt concentration and was combined with the original CEE. The CEE was passed through a 0.45m filter for sterilization, and the protein concentration was determined using the Bradford method. Chicken embryo extract was frozen at −20°C and thawed prior to use. To determine growth rates, SC-1 CEF cells were plated at 3 × 105 cells/10-cm dish using culture conditions as described above. At 80% confluency, cells were trypsinized and counted, and the number of population doublings per day was calculated. The CEE was first added to SC-1 cells at passage 45. At passage 80, CEE was withdrawn from a subset of cells.
Morphological Analysis Images of live cells were captured using phase-contrast microscopy (100×). Morphological analysis of the cells was accomplished by comparing cell size.
RT-PCR Analysis For semiquantitative RT-PCR, 3 g of RNA treated with DNase I was converted to cDNA using Superscript II RT3 by following the manufacturer’s instructions. A portion (1 L) of the RT reaction was used to amplify cDNA fragments with chicken-specific primers (Table 1). All semiquantitative cDNA fragments were amplified using TaKaRa Ex Taq.4 The PCR products were verified to be in the linear range and were visualized by ethidium bromide staining. To validate the above quantitation method, PCR products also were amplified for 15 cycles (undetectable by ethidium bromide staining) and were detected by standard Southern blot hybridization using their corresponding [α-32P]-labeled cDNA probes prepared by RT-PCR (data not shown). Images were processed using the Eagle Eye II still video system.5 A minimum of 3 independent RT-PCR experiments were analyzed using the NIH image software program6 and normalized using the expression levels of gylceraldehyde-3-phosphate dehydrogenase (GAPDH).
Statistical Analyses Serial data generated from cells were analyzed statistically to determine treatment effects using Proc GLM in a two-way ANOVA for repeated measures (SAS/
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tion. Most have been immortalized via viral or chemical methods (Akiyama et al., 1973; Nazerian and Witter, 1975; Dinowitz, 1977; Nazerian et al., 1977; Kaaden et al., 1982; Gionti et al., 1989; Barald et al., 1997; Moscovici et al., 1977), providing a significant obstacle for molecular analysis. The recently developed spontaneously immortalized SC-1 cell line is free of endogenous viruses and is not transformed. However, the decreased growth rate has been a concern in terms of the line’s utility. The present study describes an attempt to increase the growth rate and improve the morphological characteristics of the spontaneously immortalized SC-1 CEF cell line. A critical requirement for the growth of cells in culture is the presence of appropriate growth factors. In the case of mammalian hemopoietic cells, these activities have been extensively investigated (see Clark and Kamen, 1987), and many growth factors have been identified, sequenced, and produced recombinantly. Chicken embryo extract (CEE) contains many growth factors that stimulate cell growth. This is evidenced by the fact that after fertilization, a chicken develops in only 21 d with all necessary growth factors provided in the egg. It was hypothesized that addition of CEE to standard growth medium might enhance the growth rate of the SC-1 cell line. The growth rates of mouse neural crest cells and human muscle cells have been shown to increase when CEE is present in the culture medium (Maxwell et al., 1996; Yasin et al., 1981). The number of growth factors identified in CEE increases constantly, and the combinatorial needs of cells grown in culture are being investigated. In the present study, CEE was isolated and used to determine whether the growth rate and morphology of the spontaneously immortalized SC-1 cell line could be enhanced. Analysis of genes involved in the p53 and Rb pathways was performed using reverse transcription (RT)-PCR to elucidate the mechanisms by which CEE may alter the expression of important cell cycle regulatory genes.
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EMBRYO EXTRACT ENHANCES GROWTH AND MORPHOLOGY TABLE 1. Gene primers used in this study Gene
Accession number
Primers1
Annealing temperature
NM_205264
F5′-CCGTGGCCGTCTATAAGAAA-3′ R5′-GCAGGAACTGTTGCACATGA-3′
60°C
MDM2
AF005045
F5′-CAATCAACAAGACTCTACGCTGGCTG-3′ R5′-TCATCTTCATCTGTGAGCTCCTGTCC-3′
65°C
p21
NM_204396
F5′-ACTTCAACTTCGAGACCGAGAC-3′ R5′-GGGCTCTTCCTATACATTGCAC-3′
58°C
p27
NM_204256
F5′-CATGCCAGAGGAAGTGGAAT-3′ R5′-TTCGGCCTACACAGTGAGTG-3′
55°C
Rb
AF323706
F5′-GGAGTCCAAGTTCCAACAAAAG-3′ R5′-ACAAACAAACACAGTGTCCCAG-3′
60°C
p15INK4b
NM_204433
F5′-GGGATTAGAGGGATGTGTGG-3′ R5′-GGCAGGAGGTGTTTACCAAA-3′
60°C
E2F-1
NM_205219
F5′-CCTCACCTCACCCATCCCTACCC-3′ R5′-GTGACAGTGCAATGAACTCATCCGC-3′
60°C
ARF
NM_204434
F5′-CCCGTAGAGCTTTGCAGTTC-3′ R5′-ACATGCAGGAACGGATCTTC-3′
60°C
1
F = forward; R = reverse.
STAT7). Differences in mRNA expression for each gene were determined using Proc GLM in a one-way ANOVA. Probabilities ≤ 0.05 were considered significant.
RESULTS Growth Rate and Morphological Analysis The morphology of the SC-1 cells was compared with primary CEF cells, immortal HCEFi and BCEFi cells, and the DF-1 cell line (the only other RT-negative spontaneously immortalized chicken cell line to have been established). The immortal DF-1 (passage 262), BCEFi (passage 120), and HCEFi (passage 96) had similar morphologies. These immortal cells appeared to be smaller and more uniform than primary passage 6 CEF cells, which displayed a spindle-shaped morphology, which are phenotypical of fibroblasts (Figure 1a). Immortal SC1 cells (passage 125) were elongated and possessed a high cytoplasmic:nuclear volume ratio, indicative of early senescent cells (Figure 1a). Treatment of the primary CEF cells and immortal DF1, HCEFi, and BCEFi cells with varying concentrations of CEE resulted in no phenotypical differences. Similarly, treatment of the SC-1 cells with 10 or 25 g of CEE/mL resulted in no phenotypical differences compared with untreated SC-1 cells. However, SC-1 cells treated with 50 g of CEE/mL were smaller, had a lower cytoplasmic:nuclear volume ratio than untreated cells, and were more spindle-shaped, characteristic of fibroblasts. The CEE-treated SC-1 cells at 50 g/mL also tended to display more contact (arrow in Figure 1a) with each other compared with untreated SC-1 cells.
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SAS/STAT, 1990, Version 9.1, SAS Institute Inc., Cary, NC.
The average growth rate of the primary CEF cells was 1.00 ± 0.13 population doublings per day (PDD), whereas the average growth rate of the SC-1 cells from passage 120 to 122 was only 0.60 ± 0.07 PDD (P < 0.05). The growth rates of the other immortal cells also were greater than that of the SC-1 cells with the DF-1 cells averaging 1.38 ± 0.10 PDD from passages 262 to 274, the HCEFi averaging 1.00 ± 0.09 PDD from passages 96 to 108, and the BCEFi averaging 1.22 ± 0.10 PDD from passages 120 to 132 (P < 0.05; Figure 1b). After 3 yr in culture, the growth rate of the SC-1 cells had increased from 0.20 PDD at passage 40 to 0.60 PDD at passage 107, which has been maintained to the current passage 160. Various concentrations of CEE were tested to determine which, if any, would increase growth rate. The concentrations of CEE tested ranged from 0 to 500 g/mL. In primary cells, concentrations of CEE > 100 g/mL caused cells to senesce earlier than untreated cells (P < 0.05; Figure 2a). In all immortal cells, concentrations of CEE ≥ 100 g/mL inhibited growth (data not shown). Addition of CEE to DF-1, BCEFi, and HCEFi cells at < 100 g/mL did not alter the growth rate of the cells. Chicken embryo extract was first added to the SC-1 cells at passage 45. By using a repeated measures test, addition of 10 or 25 g of CEE/mL appeared to have no significant effect on the growth rate of the SC1 cells, even after 80 passages in culture (P > 0.05; Figure 3a). However, addition of 50 g of CEE/mL significantly enhanced the growth rate of the SC-1 cells after 80 passages in culture (P < 0.05; Figure 3b). The average growth rate increased from 0.60 ± 0.07 PDD without CEE to 0.78 ± 0.06 PDD with 50 g of CEE/mL (P < 0.05). After pretreatment of the SC-1 cells with 50 g of CEE/mL for 40 passages, the CEE was withdrawn. After withdrawal of CEE for 40 passages, the growth rate of SC1 cells remained elevated compared with untreated SC-
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p53
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FIGURE 1. (a) Representative photomicrographs of primary passage 6 chicken embryo fibroblast (CEF) cells and immortal passage 262 DF-1 cells, passage 125 SC-1 cells, and passage 125 SC-1 cells + 50 g of chicken embryo extract (CEE)/mL. Bars represent 100 m. The arrow indicates increased cell contact. (b) Average growth rate of primary CEF (passages 4 to 12) and immortal SC-1 (passages 120 to 132), DF-1 (passages 262 to 274), breast-derived CEF immortal line (BCEFi; passages 120 to 132), and heart-derived CEF immortal cell line (HCEFi; passages 96 to 108) cells. Bars indicate SD. a-cBars with different letters indicate significant differences.
1 cells (Figure 3c). The growth enhancement that was observed in the SC-1 cells that had the CEE withdrawn continues to date.
mRNA Expression Analysis Because high levels of CEE (≥100 g/mL) caused early senescence in primary CEF cells, mRNA expression lev-
els of various cell cycle regulatory genes were measured. In primary CEF cells, levels of p53 (68 ± 4%), p16INK4a alternate reading frame sequence (ARF; 67 ± 3%), p15INK4b (87 ± 4%), Rb (60 ± 3%), and E2F-1 (76 ± 3%) mRNA expression were increased (P < 0.05) with addition of greater concentrations of CEE (100 g/mL), whereas expression of MDM2, p21, and p27 mRNA were decreased 30 ± 5% (P < 0.05; Figure 2b).
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duction in the expression of E2F-1 mRNA that was maintained after withdrawal of the CEE for 40 passages (Figure 4).
DISCUSSION
In the SC-1 cells, levels of p53, MDM2, p21, and p27 mRNA were decreased compared with primary CEF cells (S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data) and did not change significantly with varying concentrations of CEE or upon removal of CEE (Figure 4). The levels of ARF mRNA in the SC-1 cells were twice that measured in primary CEF cells (S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data). Increasing amounts of CEE decreased the expression of ARF mRNA in the SC-1 cells with 50 g of CEE/mL resulting in a 3-fold decrease in ARF expression compared with untreated SC-1 cells. The reduction in ARF mRNA expression in the presence of 50 g of CEE/mL was maintained even after withdrawal of the CEE for 40 passages. In SC-1 cells, the levels of Rb and p15INK4b were unchanged with varying concentrations of CEE (data not shown), whereas increasing concentrations of CEE caused a re-
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FIGURE 2. (a) Growth curve of primary chicken embryo fibroblast (CEF) cells with and without 100 g of chicken embryo extract (CEE)/ mL. The cells were thawed at passage 2 and then plated at a density of 3 × 105 cells/10-cm dish. At 80% confluency cells were trypsinized and counted, and the number of population doublings per day was calculated. (b) Relative mRNA expression levels of cell cycle regulatory genes for passage 6 CEF cells (− and + 100 g of CEE/mL). For each gene analyzed, the expression level was set to 1.00 for passage 6 CEF cells that had not been treated with CEE. The levels of the sample that had been treated with 100 g of CEE/mL were adjusted accordingly. A minimum of 3 independent reverse transcription-PCR experiments were analyzed using the NIH image software program (ImageJ 1.24O, 2001, NIH, Washington, DC) and normalized using the expression levels of gylceraldehyde-3-phosphate dehydrogenase. Differences ≤ 0.05 were considered significant.a,bBars with different letters indicate significant differences.
In the present study the spontaneously immortalized SC-1 cell line was treated with CEE in an attempt in increase growth rate and improve morphology. Because the availability of nonvirally and nonchemically derived cell lines is limited, creation of such cell lines is invaluable to provide suitable substrates for cellular and molecular analysis and for virus propagation and vaccine production. In fact, a pharmaceutical company has propagated Marek’s virus in the SC-1 cells and shown that titers were similar to those in primary CEF and immortal DF-1 cells (data not shown). The SC-1 cell line is only the second RT-negative spontaneously immortalized CEF cell line that has been reported. However, its slow growth rate has been a limitation to its utility as a cellular or molecular reagent. In an attempt to increase the growth rate of the SC-1 cell line to a rate that was more similar to primary CEF cells or other immortal cells (DF1, HCEFi, and BCEFi), the SC-1 cell line was treated with CEE. It was discovered that treatment of any of the cells with ≥100 g of CEE/mL restricted growth, suggesting that high concentrations of factors present in CEE are inhibitory to the growth of primary and immortal CEF cells. The primary CEF cells and immortal DF-1, HCEFi, and BCEFi cells did not benefit from the addition of CEE to the basal medium, indicating that these cells did not require additional growth factors present in CEE for optimum growth. On the other hand, the slower growth rate of the SC-1 cells suggested that the lack 1 or more growth factors was constraining the growth rate of the cells. To this end, the SC-1 cells were treated with CEE in an attempt to attain a growth rate that was more similar to the growth rate of primary CEF and other immortal CEF cells. In fact, the growth rate of the SC1 cells was enhanced by treatment with 50 g of CEE/ mL. It appeared that this concentration of CEE provided the appropriate concentration of growth factors that were beneficial for the growth of SC-1 cells and were deficient in basal media. The population of SC-1 cells was not as uniform as the primary CEF cells or the immortal DF-1, HCEFi, or BCEFi cells. The DF-1, BCEFi, and HCEFi cell lines had a smaller and more compact morphology than primary CEF or SC-1 cells. The SC-1 cells were more similar to primary CEF cells than were any of the other immortal cells examined. However, the SC-1 cell line was composed of some flattened and enlarged cells that had an increased cytoplasmic:nuclear volume ratio. Treatment of the SC-1 cells with 50 g of CEE/mL reduced the number of these flattened and enlarged cells, suggesting that this concentration of CEE was most beneficial in improving the morphology of the SC-1 cell line. Treatment of the primary CEF cells and immortal DF-1,
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FIGURE 3. (a) Growth analysis of SC-1 cells (passages 125 to 142) with varying concentrations of chicken embryo extract (CEE). (b) Growth analysis of the SC-1 cells (passages 125 to 142) with and without 50 g of CEE/mL. (c). Growth analysis of SC-1 cells (passages 125 to 142) + 50 g of CEE/mL and SC-1 cells that had been pretreated with 50 g of CEE/mL for 40 passages and then had the CEE withdrawn for 40 passages. Serial data generated from cells were analyzed statistically to determine treatment effects using Proc GLM in a 2-way ANOVA for repeated measures (SAS/STAT, 1990, Version 9.1, SAS Institute Inc., Cary, NC). Differences ≤ 0.05 were considered significant.
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BCEFi, and HCEFi cells with varying concentrations of CEE did not alter the morphology of these cells. Again, this finding suggested that these cells did not require additional growth factors deficient in basal media. An unexpected observation was that treatment of primary CEF cells with ≥100 g of CEE/mL prompted early onset of senescence. To gain a better understanding of this phenomenon in primary CEF cells, the mRNA expression levels of various cell cycle regulatory genes were examined via RT-PCR. The tumor suppressor p53 initiates pathways that result in cell senescence or death (Sherr and DePinho, 2000). As expected, levels of p53 mRNA were increased in primary CEF cells treated with ≥100 g of CEE/mL. Another important regulatory gene in the p53 pathway is MDM2, a proto-oncogene that is elevated in 30 to 40% of human tumors (Oliner et al., 1992). The MDM2 gene product binds to p53, inactivating its transcriptional activity (Momand et al., 1992; Oliner et al., 1993). Therefore, it would be expected that MDM2 should be decreased in senescent cells. As predicted, levels of MDM2 were decreased in primary CEF cells that were treated with concentrations of CEE ≥ 100 g/mL, again characteristic of senescent cells. The p21 (El-Diery et al., 1993; Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993a) and p27 (Polyak et al., 1994; Slingerland et al., 1994) genes belong to the Kip/ Cip family of CDK-inhibitory proteins that block transition from the G1 to the S phase of the cell cycle. In normal cells, DNA damage causes cell cycle arrest at the G1/S phase of the cell cycle via p53 stimulation of p21 expression (El-Deiry et al., 1993, 1994). It has been shown that levels of p21 and p27 are highest at senescence (Noda et al., 1994) and that over-expression induces cell cycle arrest at the G1/S phase of the cell cycle (El-Deiry et al., 1993, 1994; Polyak et al., 1994; Toyoshima and Hunter, 1994). However, in the present study, the levels of p21 and p27 were decreased in primary CEF cells that had been treated with ≥100 g of CEE/mL. It is possible that elevated levels of p53 initiated other pathways to trigger early senescence in the CEF cells that were treated with ≥100 g of CEE/mL. The ARF gene product functions upstream of MDM2 and p53 by stably binding to MDM2, thus preventing MDM2 from exporting p53 to the cytoplasm for degradation (Tao and Levine, 1999; Weber et al., 1999; Zhang and Xiong, 1999). In fact, it has been reported that ARF is required for senescence and is inactivated in many human tumors (Kamijo et al., 1997). Here, as expected, it was observed that levels of ARF mRNA were increased in primary CEF cells treated with ≥100 g of CEE/mL that entered senescence early. Another important gene that is involved in senescence is p16INK4a. Because chickens encode p15INK4b in place of p16INK4a (Kim et al., 2003), levels of p15INK4b were measured. As expected the levels of p15INK4b were elevated in primary CEF cells treated with ≥100 g of CEE/ mL that entered senescence early. It is likely that the increased expression of p53, ARF, and p15INK4b mRNA
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FIGURE 4. Relative mRNA expression levels of cell cycle regulatory genes for passage 125 SC-1 cells. The amount of chicken embryo extract (CEE; g/mL) is shown at the bottom of the figure (w50 = cells treated with 50 g of CEE/mL for 40 passages and then had the CEE withdrawn for 40 passages). For each gene analyzed, the expression level was set to 1.00 for SC-1 cells that had not been treated with CEE. The levels for other samples were adjusted accordingly. A minimum of 3 independent reverse transcription-PCR experiments were analyzed using the NIH image software program (ImageJ, 1.24O, 2001, NIH, Washington, DC) and normalized using the expression levels of gylceraldehyde-3-phosphate dehydrogenase. Differences ≤ 0.05 were considered significant. a–c Bars with different letters indicate significant differences.
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contributed to early onset of senescence in the primary CEF cells treated with ≥100 g of CEE/mL. In the SC-1 cells, varying concentrations of CEE (0, 10, 25, and 50 g/mL) did not significantly alter the expression of p53, MDM2, p21, p27, or p15INK4b. This was not surprising because expression of most of these genes were already lower than the expression in primary CEF cells (S. A. Christman, B.-W. Kong, M. M. Landry, and D. N. Foster, unpublished data). However, increasing concentrations of CEE lowered the expression of ARF and E2F-1. The expression of ARF in untreated SC1 cells was increased compared with primary CEF cells, whereas most immortal cells display reduced levels of ARF mRNA (Kamijo et al., 1997). Treatment of the SC1 cells with CEE decreased the expression of ARF mRNA to correspond to levels observed in other immortal cells (Kamijo et al., 1997). The lower levels of E2F-1 that were observed as the concentration of CEE increased were not expected. Previous work in our laboratory has shown that mRNA, protein, and functional activity of E2F-1 were dramatically elevated in all immortal cells studied (Kim et al., 2001b). However, a link between E2F-1 and ARF expression may explain the lower levels of E2F-1 observed in the SC-1 cells as the levels of ARF decreased (Bates et al., 1998). Another important finding of this study was that the increased growth rate, improved morphology, and pattern of gene expression were maintained in the SC-1 cells that were treated with 50 g/mL for 40 passages and then had the CEE withdrawn for 40 passages. This result suggested that treatment of the SC-1 cells with the appropriate concentration of CEE (50 g/mL) provided the cells with growth factors that altered gene expression to allow the SC-1 cells to improve their growth rate and enhance their morphology and that this improvement was imprinted and retained upon CEE withdrawal. In conclusion, it appears that treatment of the slowergrowing SC-1 cell line with CEE was successful in increasing the growth rate and improving the morphology of the cells and that the improvements achieved through CEE treatment were retained even after CEE withdrawal. This progression of the SC-1 cell line should aid in its utility as an appropriate model for investigations into cell cycle regulation and virus propagation.
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