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The Rowett Research Institute, Aberdeen, AB21 9SB Scotland, UK
2To whom correspondence should be addressed. E-mail: wdr{at}rri.sari.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • mice • cell growth • growth arrest • apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,2
). Abnormal growth during these early stages can have long-lasting effects on the offspring (3
). Studies in vitro have shown that the development of preimplantation embryos is augmented by the addition of indispensable and dispensable amino acids to culture media (4
). In vivo, the balance of amino acids available for the growth of fetal cells is determined by the maternal diet and the metabolism of the dam. In addition to distortions caused by inadequacies in the maternal diet, there is also evidence to suggest that the demand for dispensable amino acids may exceed the capacity of the synthetic reactions, further imbalancing the amino acid supply (5
). The contribution of dispensable amino acids to the regulation of embryonic and fetal growth is unknown, but two different mechanisms are possible, i.e., a shortage of amino acids limiting protein synthesis or a shortage of nucleotides limiting DNA synthesis.
Mammalian cells react to nutrient deficiencies by altering their patterns of gene expression, and there is evidence that the nature of the response depends on whether the cells are subject to amino acid or nucleotide deficiency. For example, levels of the gadd153 gene mRNA (also known as CHOP-10 or ddit3) increase by severalfold after mammalian cells are transferred to a culture medium deficient in an essential amino acid (6
–8
). However, a similar degree of growth arrest caused by blocking nucleotide synthesis with an inhibitor does not induce gadd153 expression (9
,10
). The mRNA for gadd153 is just one of a diverse group of genes that are overexpressed by nutrient-stressed cell cultures (11
,12
). These genes also play an important part in normal fetal development and are expressed at high levels in most fetal tissues (13
). Nutrient-mediated changes in the patterns of mRNAs in cells have the potential to change fetal growth and development, permanently altering the subsequent physiology of the adult.
The small quantities of mRNA present in preimplantation embryos and the complex array of different cell types found in the developing fetus make it difficult to characterize specific changes in mRNAs resulting from an interruption or deficiency in the nutrient supply. The stem cells of malignant teratomas or teratocarcinomas offer a convenient alternative. These embryonal carcinoma cells have been widely used to study various aspects of embryogenesis because they have properties similar to those of the inner cell mass and primitive endoderm cells of the embryo (14
). Using this system, specific nutrient stresses can be applied to cells either by reducing the concentration in the culture medium or by the addition of specific inhibitors. The indispensable amino acid lysine was chosen to investigate the effect of amino acid deficiency because this amino acid does not enter into intermediary metabolism and is primarily a substrate for protein synthesis in F9 cells. The synthesis of nucleotides from amino acids can be blocked by treating cells with phosphonoacetyl L-aspartic acid (PALA).3
This compound is a specific inhibitor of aspartate carbamyl transferase, a key step in the de novo synthesis of pyrimidine nucleotides from aspartic acid (15
,16
). Using these two different models, which involve different mechanisms of growth arrest, RNA extracted from the cells was screened for genes known to be associated with growth arrest. In addition, we identified additional genes associated with growth arrest by comparing the patterns of expression using differential display reverse transcription-polymerase chain reaction (DDRT-PCR) (17
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mouse F 9 embryonal carcinoma cells (CRL-1720, American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s essential medium containing 10% fetal calf serum (Gibco, Paisley, UK). Serum from the same batch was used for all experiments. Lysine-deficient medium was prepared according to the same formula except that lysine was omitted. Serum-dependent growth arrest was achieved by replacing the fetal calf serum with 2% newborn calf serum. The culture of F9 cells as monolayers on gelatin-coated plates was described previously (7
). Cell growth was measured by staining cells with Crystal violet as described previously by Gilles et al. (18
). Cells were induced to differentiate by treating them with 10-4 mol/L all-trans-retinoic acid. PALA was a generous gift from Dr. J. Johnson, Drug Synthesis Branch, National Cancer Institute, Bethesda, MD.
Northern analysis.
The isolation of total RNA, its separation on 1.2% agarose gels, transfer to a nylon membrane and hybridization have been described previously (7
). The probes for gadd153 and for ß-actin were derived from the plasmids pA5A4 (a gift from A. J. Fornace, NIH) and pMP90 (Dr. J. Hesketh, University of Newcastle, UK). Probe templates were labeled with [
-32P]-dCTP using a Megaprime labeling kit (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). After hybridization, the blots were washed in 0.5X SSC + 1% SDS at 65°C and quantified by imaging on a wire proportional counter (Packard Instant Imager, Packard Biosciences Pangbourne, Bucks., UK). Errors in loading and transfer were corrected by reprobing the blots for 18S ribosomal RNA.
DDRT-PCR.
This was performed essentially as described by Liang and Pardee (17
). Briefly, RNA (50 µg) was dissolved in 20 µL buffer [20 mmol/L Tris-HCl (pH 8.4), 2 mmol/L MgCl2, 50 mmol/L KCl] containing 1 U DNaseI (Gibco). The sample was incubated at 25°C for 10 min, precipitated with isopropanol, washed in 70% ethanol and resuspended in DEPC-treated water. DNase-treated RNA (10 µg) was dissolved in 20 µL of RT buffer containing 20 µmol/L dNTP, 2.5 mmol/L HT11X primer (AAGCTTTTTTTTTTTX, where X = A, G, or C), 10 mmol/L dithiothreitol, 40 U RNase block (Stratagene, Cambridge, UK), and 200 U of AMV reverse transcriptase (Superscript, Gibco). The reaction mixture was incubated at 37°C for 1 h and heated to 95°C for 5 min. RT reactions were performed in duplicate. Blanks, one containing no enzyme, the other omitting the template RNA were set up at the same time to check for contamination.
Table 1
shows the sequences of the DDRT primers, which were synthesized by Cruachem (Glasgow, UK). The PCR reaction was carried out in a 50 µL volume of PCR buffer containing 20 µmol/L dNTP, 9.25 kBq [
-32P] dCTP, 25 µmol/L HT11X primer, 25 µmol/L forward arbitrary primer (Table 1)
, and 2 µL of RT mixture. The reactions were initiated by the addition of 2.5 U of Taq DNA polymerase (Roche Diagnostics, Lewes, East Sussex, UK) and the PCR was carried out for 40 cycles. Each cycle comprised denaturation at 94°C for 30 s, annealing at 61°C for 45 s and extension at 72°C for 45 s. Products were analyzed on 6% acrylamide wedged sequencing gel containing 7 mol/L urea in the running buffer. After autoradiography, bands showing differential expression were cut out with a clean scalpel blade and allowed to rehydrate in 50 µL H2O overnight at 37°C. An aliquot was used as a template for reamplification using the same primers. The PCR product was purified from a 1% agarose gel, reamplified with Pfu DNA polymerase and cloned into the Srf I site of the pCR-Script SK(+) plasmid using the pCR-Script Cloning Kit (Stratagene, Cambridge UK). The plasmid insert was sequenced using an ABI 377 DNA sequencer.
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Northern analysis was carried out on the RNA extracted from three separate cultures. The experiment was repeated three times and the means from each experiment were used in the final calculations with n = 3. Experimental means were compared with the control using unpaired Student’s t test and were considered significantly different at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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shows the logarithmic growth curves of F9 teratocarcinoma stem cells cultured in media containing different concentrations of lysine and PALA. Cells in complete medium containing 10% fetal calf serum divided with a doubling time of 
15 h. The concentration of lysine that supported 50% of this growth was calculated to be 57 µmol/L (Fig. 1
a). A similar analysis showed that 90 µmol/L PALA inhibited growth by 50% (Fig. 1
b).
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). To achieve approximately similar degrees of growth arrest, the medium contained either 19 µmol/L lysine or 120 µmol/L PALA. Both of these treatments increased the doubling time to 70 h. After 24 h of culture in lysine-deficient medium, the levels of gas5 and gadd153 mRNAs were increased by between 5- and 15-fold compared with the normally growing controls, consistent with previous results (7
). The response of gadd153 in cells treated with PALA was much smaller with only a twofold increase (P < 0.001). There was a significant decrease in gas5 mRNA in the PALA-treated cells with the levels falling to approximately half of those in the control cells. In an attempt to identify genes up-regulated after the inhibition of nucleotide synthesis, two genes associated with the inhibition of DNA synthesis were also studied. Levels of p53 and gadd45 mRNAs were increased between two- and threefold in cells arrested by both amino acid and nucleotide deficiency.
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). Several of the other clones did not show increases in expression when subjected to a more comprehensive analysis. All of the bands that appeared to be differentially expressed in PALA-treated cells were found to be false positives. The GenBank and EMBL databases were searched using the BLAST protocol to identify sequences homologous to the positive clones. The results of this search are summarized in Table 2
.
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5 kb. The levels of this message were two- to fourfold higher in lysine-deficient cells than in control cells. Treating cells with PALA had no effect on its expression. Analysis of the time course of expression (Fig. 4a
) showed that the gene was induced over a 24-h period and remained elevated throughout this period. When cells were induced to differentiate by retinoic acid treatment, an increase in the expression of the hoxB2 gene confirmed the induction of differentiation (7
). The mRNA was not induced during differentiation and the induction by lysine deficiency was not affected (Fig 5a
).
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). This clone is represented in a number of mouse expressed sequence tag (EST) sequences in the UniGene Cluster Mm.36715 and is moderately similar to the cDNA for human polyamine-induced factor (PA-1) (20
,21
). The probe hybridized to a 1.8-kb message, which is similar in size to that reported for PA-1. The time course shown in Figure 4
b shows that the mRNA was induced by one- to twofold in lysine-deficient cells. In differentiating cells, there was a similar response to lysine deficiency with an increase of three- to fourfold (Fig. 5
b). Under both conditions, the mRNA levels reached a maximum within 24 h after transfer to the lysine-deficient medium but declined thereafter.
The 730-bp fragment designated clone 5 showed 94% identity over 668 bp with the mouse doublesex-related gene (Dmrt1) cDNA. The probe hybridized to a message of 2.5 kb, which is close to the size of the Dmrt1 message, 2.2 kb (22
). The mRNA was induced by 10- to 40-fold in lysine-deficient cells. Analysis of the time course of expression showed that the gene was induced over a 24-h period and remained elevated (Fig. 4
c). Treating cells with retinoic acid did not induce the mRNA and did not affect its induction by lysine deficiency (Fig. 5
c).
The 480-bp fragment designated clone 7 showed 95% identity over 463 bp with a number of mouse EST sequences in the UniGene Cluster Mm.41305 (2410136Rik) but could not be identified. The probe hybridized to a message of 3.3 kb, which was induced by two- to fourfold in lysine-deficient cells. The gene was induced over a 24-h period and remained elevated throughout this period (Fig. 4
d). Treating cells with retinoic acid did not induce the mRNA but markedly reduced its induction by lysine deficiency (Fig. 5
d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Therefore, these experiments have investigated a random sample taken from the mRNA pool and do not represent a comprehensive analysis of gene expression. It is still possible that some mRNAs are highly up-regulated in the PALA-treated cells. It is also likely that other amino acid sensitive genes remain to be identified.
This is the first report to show that Dmrt1 is a growth arrest gene. The gene, related to worm and fly sexual regulators, is required for the development of the mammalian testis. In most vertebrates, it is expressed in the genital ridges of both sexes and then becomes sex specific and confined to the testis at the end of gonadogenesis (22
,24
). Analysis of mutants shows that Dmrt1 is required for multiple aspects of testis differentiation and is not required for ovary development (25
). The high level of expression is therefore not so surprising given the testicular origin of F9 cells. The product of Dmrt1 contains a zinc finger DNA binding domain, which has been implicated in the regulation of development. A number of studies have suggested that undernutrition can disrupt the normal development of the gonads (26
). Because the Dmrt1 transcript is growth sensitive, it may play an important role in mediating nutritional effects on testis development.
The mRNA for polyamine-modulated factor L-1 is also up-regulated by amino acid deficiency. As a member of the b-zip family of transcriptional activators, PA-1 is related to gadd153, which is also overexpressed under conditions of amino acid deficiency (21
). A dimer formed between PA-1 and the transcription factor Nrf-2 through the leucine zipper motif regulates the polyamine-induced transcription of spermine/spermidine N-acetyl transferase (20
). Depletion of polyamines or alterations in their metabolism arrest the cell cycle and can induce apoptosis. Because amino acids are the precursors for polyamine synthesis, the regulation of mRNA levels by the amino acid supply may be important in the regulation of polyamine production.
The functions of the remaining EST are not known, although both are well represented in libraries from fetal and embryonic tissues. It is likely that at least one of these is also important during development because it has been identified as differentially expressed in the fetal mouse. Indeed, it is striking that many of the mRNAs for genes associated with amino acid deficiency and growth arrest are present at high levels in the fetus (13
). This suggests that the products of these genes play an important role during development. It remains to be seen whether nutrient-mediated changes in the steady-state levels of mRNAs are of physiologic importance for fetal development. The genes regulated by amino acid deficiency may be important in preventing tissue damage by inducing apoptosis to eliminate damaged or defective cells (9
,27
,28
). It is possible that the other inducible mRNAs such as PA-1 are also part of this apoptotic signal.
In general, the majority of the changes observed were positive increases in mRNA levels. A few bands that appeared to be down-regulated were observed in DDRT gels; however, all of those that were tested proved to be false positives. This suggests that down-regulation of gene expression is a relatively rare event. The only exception to this was gas5, whose levels fell in PALA-treated cells. This may reflect the fact that gas5 codes for a catalytic RNA rather than a protein (29
,30
). The gas5 RNA plays an important role in ribosomal RNA processing and therefore a fall in RNA turnover may be reflected in a reduction in ribosome synthesis. As a measure of rRNA processing, gas5 may be a useful marker for changes in ribosome turnover when cell proliferation is interrupted.
There are a number of similarities in the patterns of expression and induction of gadd153, gas5, PA-1, and Dmrt. This suggests that there are some common components in the pathways controlling the response to amino acid deficiency. When cells in culture are deprived of a single amino acid there is a reduction in general protein synthesis regulated primarily through the phosphorylation of eukaryotic initiation factor (eIF) 2 and the inhibition of eIF2ß (31
). Some mRNAs, however, are exempt from this regulation and continue to be translated by a cap-independent mechanism (32
). The continued protein synthesis stabilizes the mRNA and increases its levels relative to other mRNAs (33
). An analysis of the gadd153 promoter and that of other amino acid-regulated genes has shown that a stretch of nine nucleotides is required for the response to amino acid starvation, and mutations result in a complete loss of responsiveness (34
,35
). It will be interesting to see which of these mechanisms are involved in the regulation of gas5, Dmrt1, PA-1, and the EST clones.
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FOOTNOTES |
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3 Abbreviations used: DDRT-PCR, differential display reverse transcriptase-polymerase chain reaction; EIF, eukaryotic initiation factor; EST, expressed sequence tag; PA-1, polyamine-induced factor; PALA, phosphonacetyl-L-aspartic acid.
Manuscript received 1 February 2002. Initial review completed 19 March 2002. Revision accepted 13 May 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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