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Originally published In Press as doi:10.1074/jbc.M308451200 on September 23, 2003

J. Biol. Chem., Vol. 278, Issue 50, 49868-49873, December 12, 2003
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Methylthioadenosine Phosphorylase Regulates Ornithine Decarboxylase by Production of Downstream Metabolites*

Ahmad L. Subhi{ddagger}, Paula Diegelman§, Carl W. Porter§, Baiqing Tang{ddagger}, Zichun J. Lu¶, George D. Markham¶, and Warren D. Kruger{ddagger}||

From the Divisions of {ddagger}Population Science and Basic Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 and the §Roswell Park Cancer Institute, Pharmacology and Therapeutics Department, Buffalo, New York 14263

Received for publication, August 1, 2003 , and in revised form, September 11, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639–6644). Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo. In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity. We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1{Delta} cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools. Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines. The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis. Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels. Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A quarter century ago, Toohey (10) first recognized that certain murine malignant hematopoietic cell lines lacked methylthioadenosine phosphorylase (MTAP)1 activity. MTAP is a key enzyme in the methionine salvage pathway (see Fig. 1). This pathway functions to salvage methylthioadenosine (MTA), which is formed as a by-product of polyamine metabolism. Phosphorolysis of MTA by MTAP results in the conversion of MTA into adenine and methylthioribose 1-phosphate. A series of reactions then salvages the methyl-thio group from methylthioribose 1-phosphate to form methionine. This pathway has been most extensively studied in Klebsiella pneumoniae (11–14) but has also been shown to exist in rat liver (15–17) and in Saccharomyces cerevisiae (18, 19).



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  		FIG. 1.
Methionine salvage and polyamine pathways. Enzymes are shown in italic type. Yeast genes are shown in parenthesis. SAMDC, S-adenosylmethionine decarboxylase; dSAM, decarboxylated S-adenosylmethionine.

 
Loss of MTAP activity through gene deletion is common in many kinds of human cancers including non-small cell lung cancer, glioma, T-cell acute leukemia, bladder cancer, osteosarcoma, and endometrial cancer (2–5, 20–22). Recently, we found that expression of MTAP in an MTAP-deleted breast adenocarcinoma cell line (MCF-7) resulted in a dramatic inhibition of tumorigenicity in vitro and in vivo, showing that MTAP can function as a tumor suppressor gene (1). We also found that MTAP expression causes a significant decrease in intracellular polyamine levels and alters the ratio of putrescine to total polyamines. Consistent with this observation, the polyamine biosynthesis inhibitor {alpha}-difluoromethylornithine inhibits the ability of MTAP-deficient MCF-7 cells to form colonies in soft agar, whereas the addition of the polyamine putrescine stimulates colony formation in MTAP-expressing MCF-7 cells. These results indicate that the tumor suppressor activity of MTAP may be mediated by its effect on the intracellular polyamine pools.

Polyamines are small, aliphatic amines involved in a variety of cellular processes including transcription and apoptosis (8). The rate-limiting enzyme in the production of polyamines is ornithine decarboxylase (ODC). Elevated ODC activity has been observed in a wide variety of human and animal tumors, and overexpression of ODC in NIH/3T3 cells is sufficient to cause transformation in vitro (7, 23). Transgenic mice overexpressing ODC in skin develop skin tumors at a high frequency (9, 24). These observations show that overexpression of ODC is tumorigenic.

Examination of the S. cerevisiae genome for MTAP homologues suggests that the MEU1 gene, with 35% identity and 53% similarity over 275 amino acids, may encode the yeast MTAP homologue. Furthermore, recent studies show that cells lacking MEU1, in combination with a mutation that allows yeast to take up methylthioadenosine, are unable to grow on medium containing MTA as the sole sulfur source (25). MEU1 was initially identified in a screen for genes that regulate expression of the yeast ADH2 gene (26). Overexpression of MEU1 increased ADH2 expression, whereas deletion of MEU1 resulted in reduced ADH2 expression. At the time these experiments were published, the MTAP gene had not yet been identified, so the relationship between MEU1 and MTAP was unknown. These experiments demonstrate that MEU1 regulates ADH2 expression and indicate a link between MEU1 and gene regulation.

In this paper, we show that MEU1 encodes yeast MTAP, and we characterize the effect of loss of MTAP in Saccharomyces cerevisiae and human tumor cells. Our findings show that there is a regulatory link between the methionine salvage pathway and the polyamine pathway. Specifically, the penultimate metabolite, 4-methylthio-2-oxobutanoic acid (MTOB), acts to repress ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. These results explain why MTAP deletion causes altered polyamine profiles, and suggest that the tumor suppressor activity of MTAP may be due to its effect on ODC activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid pMEU1 Construction—The yeast MEU1 ORF was PCRamplified from total yeast DNA using primers MEU1–1F (5'-TCT GTG AAA CAT GTC) and MEU1–1R (5'-CAG TCC CCA AGG GGG). The resulting 1.3-kb product was then cloned into pCR2.1 (Invitrogen) and designated pCR2.1::MEU1. PCR2.1::MEU1 was digested with BamHI and NotI, and the resulting 1.3-kb insert was cloned into pRS316 (27) digested with the same enzymes.

Yeast Strains—Yeast strains used are indicated in Table I. Isogenic MEU1 and meu1{Delta} strains were created by transformation of AS3-2a (a ura3{Delta} met15{Delta} meu1{Delta}::LEU2 leu2{Delta}) with pMEU1 or empty vector pRS316. All other strains used were created by the S. cerevisiae deletion project (28). Standard yeast growth medium was used as described by Sherman (29). Polyamine-free medium was prepared as described by Balasundaram (30).


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  		TABLE I
Strain list

 
Mammalian Cells—MTAP-expressing and mutant expressing MCF-7 cells were created and grown as previously described (1). MIA PaCa-2 pancreatic adenocarcinoma cells were obtained from the ATCC and grown as previously described (31).

Yeast Growth Studies—Yeast cells were grown to stationary phase and then diluted to an OD = 0.5–1.0 at 600 nm, in the indicated medium. Cells were then grown in liquid medium with shaking at 30 °C for 48 h, at which time OD was determined.

Extract Preparation—Yeast cultures (45 ml) were grown aerobically to an OD = 0.5–1.0 at 600 nm, pelleted, washed with 0.9 ml of lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride), and resuspended in 750 µl of lysis buffer. Glass beads (0.5 mm; one-half weight of the cell pellet) were then added to make up ~50–70% of total volume. Cells were lysed by using a BeadBeater at 4 °C with maximum speed (4500 rpm) for 1 min three times and kept on ice between pulses (Biospec Products). After lysis, the tubes were centrifuged for 4 min, the supernatant was transferred to a new tube, and glycerol was added to a final concentration of 15%. The protein concentration of yeast extracts was determined by the Coomassie Blue protein assay reagent (Pierce) using bovine serum.

Mammalian cell lysates were prepared by three cycles of freezethawing at –80 °C in 20 mM KH2PO4, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride (Sigma) and 1 mM dithiothreitol (Sigma).

Western Blot Analysis—Forty µg of extract were run on premade 7% NuPage Tris-acetate SDS gels (Invitrogen) and electrotransferred to polyvinylidene difluoride membrane (Bio-Rad) using a standard protocol (32). Samples were probed with a 1:10,000 dilution of yeast antiornithine decarboxylase antiserum raised in rabbits (a generous gift from Martin Hoyt and Phil Cofino; University of California, San Francisco). Secondary antibody anti-rabbit horseradish peroxidase-linked antibody was obtained from Amersham Biosciences and used at a 1:3,000 dilution. The antibody-antigen complex was visualized on photographic film after treatment with SuperSignal West Dura Extended Duration Substrate (Pierce).

Measurement of Ornithine Decarboxylase, MTAP, and S-Adenosylmethionine Decarboxylase Activity—Ornithine decarboxylase activity was assayed by measuring the 14CO2 formed by decarboxylation of 1-14C-labeled ornithine as previously described (33). L-Carboxyl 14C-labeled ornithine (5 mCi/mmol) was obtained from Moravek Biochemical (Brea, CA). 20 µg of yeast or mammalian extract was used per reaction. Reactions were performed between three and seven times per sample, and results are presented as the average with the S.E. S-Adenosylmethionine decarboxylase activity was assayed by measuring the amount of 14CO2 released by decarboxylation of 1-14C-labeled S-adenosylmethionine as described (34).

MTAP activity in yeast extracts was determined using a spectrophotometric assay as described previously (1). One unit of MTAP activity is defined as the amount that catalyzes the formation of 1 µmol of adenine/min.

Measurement of Intracellular Polyamine Levels by Using HPLC— Yeast cells grown in synthetic complete medium were collected, protein extracts were prepared, and then 100 µl of supernatant was added to 100 µl 0.6 N perchloric acid and allowed to sit on ice for 5 min. The sample was centrifuged at 15,000 x g for 10 min. 100 µl of supernatant was dansylated and analyzed by high pressure liquid chromatography as described (35).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MEU1 Encodes MTAP in S. cerevisiae—Early studies showed that yeast extracts contain MTAP activity but did not identify the gene encoding the activity (18, 19). A BLAST search of the S. cerevisiae genome with human MTAP (NP_002442 [GenBank] ) reveals two open reading frames, YLR209C and YLR017W, with significant sequence similarity. Of the two, YLR017W (also known as MEU1) has the most similarity, with 35% identity over 275 amino acids to human MTAP. To test the possibility that MEU1 encoded MTAP, we constructed isogenic MEU1 and meu1{Delta} strains and measured MTAP activity in whole cell extracts (see "Experimental Procedures"). We found that MEU1 yeast extracts contained 0.38 units of MTAP activity, whereas extracts from meu1{Delta} strain had no detectable activity (<0.01 units). A second experiment to confirm our hypothesis involved examining the growth of isogenic meu1{Delta} and MEU1 strains with auxotrophic markers for adenine and methionine in media in which MTA had been substituted for methionine and adenine. We found that wild-type but not meu1{Delta} yeast can grow using MTA as a substitute for methionine and adenine (Fig. 2). Taken together, these observations show that MEU1 encodes yeast MTAP.



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  		FIG. 2.
Growth of MTAP+ and MTAP– yeast cells on MTA. Yeast strains AS24–1b (MEU1) and AS24–1b (meu1{Delta}) were diluted to OD = 0.05 at 600 nm in synthetic complete medium supplemented with 5 mM MTA and the standard amino acids and nucleotides (SC) except for the ones indicated at the bottom of the graph. Cells were grown for 48 h, and A600 was determined.

 
Effect of meu1{Delta} on Polyamine Pools and ODC Activity— Since MTA is a by-product of polyamine production, we suspected that cells lacking MTAP might have altered polyamine pools due to end product inhibition. Therefore, we measured the intracellular level of polyamines (putrescine, spermine, and spermidine) in an isogenic wild-type strain and in a meu1{Delta} strains using HPLC (see "Experimental Procedures"). We found that in synthetic complete media, MEU1 cells had undetectable levels of putrescine, whereas meu1{Delta} cells contained at least a 20-fold increase in putrescine (Fig. 3A). Surprisingly, we also found that spermidine levels were significantly increased and that the total polyamine content of meu1{Delta} cells was more than twice that of isogenic wild-type yeast (62 versus 28 nmol/mg).



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  		FIG. 3.
Polyamine and ODC activity in wild-type and salvage pathway mutants. A, strains of the indicated genotype were grown in synthetic complete medium to an OD of 1.0 and extracted with perchloric acid, and polyamines were measured as described under "Experimental Procedures." Putrescine was at levels below the detection limit (<0.5 nmol/pg) in three of the samples. Error bars, S.E. of two measurements. B, ODC activity of the indicated cells was measured in whole cell extracts as described under "Experimental Procedures." Error bars indicate S.E. of four measurements.

 
To explain the overall elevation in polyamine levels, we examined ODC activity in wild type and meu1{Delta} yeast. ODC is the rate-limiting enzyme in polyamine production and is regulated at the post-transcriptional level (36). We found that cell extracts from meu1{Delta} yeast had an 8-fold increase in ODC activity compared with isogenic wild-type yeast (Fig. 3B). This difference appeared to be due to elevated levels of ODC protein. We were unable to detect ODC protein by immunoblot in extracts derived from MEU1 cells as has been previously observed (37) and from ODC-deficient cells (spe1{Delta}), but we saw substantial expression in meu1{Delta} extracts (Fig. 4A, lanes 1–3). The increase in protein levels appeared to be even greater than the measured activity, suggesting that not all of the protein was active (lanes 1 and 2). We also noticed that meu1{Delta} yeast formed significantly larger colonies compared with isogenic MEU1 yeast on polyamine free-medium, suggesting that the elevated ODC is important for growth under these conditions (Fig. 4B). These results show that MEU1 represses ODC expression and activity.



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  		FIG. 4.
Steady-state ODC protein levels in salvage pathway mutants. A, ODC protein levels were examined in total extracts made from yeast of the indicated genotype by immunoblot (see "Experimental Procedures"). Yeast ODC runs at 53 kDa and is indicated by the arrow. The additional bands below and slightly above ODC are presumably ODC degradation products, since they are absent in both the wild-type (WT) and spe1 controls. B, comparison of colony size of isogenic wild-type and meu1{Delta} yeast grown on polyamine-free medium after 3 days of growth.

 
We also examined the effect of MEU1 on S-adenosylmethionine decarboxylase activity. We found that extracts from meu1{Delta} cells had twice the S-adenosylmethionine decarboxylase activity of isogenic MEU1 (7.7 ± 0.3 versus 14.6 ± 0.8 nmol/mg/h). This result is consistent with the idea that MEU1 acts to repress polyamine synthesis.

SPE2 and MEU1 Regulate ODC through Different Mechanisms—Previous work has shown that mutations in SPE2, encoding S-adenosylmethionine decarboxylase, also cause a dramatic increase in ODC protein levels (36) (also see Fig. 3A, lane 4). It was also shown that the ODC overexpression observed in a spe2 mutant could be suppressed by the addition of spermidine to the medium. We compared ODC expression in spe2{Delta} and meu1{Delta} strains in the presence and absence of spermidine. meu1{Delta}, spe2{Delta}, and meu1{Delta}/spe2{Delta} double mutants were grown in polyamine-free medium with or without the addition of exogenous spermidine. When we measured ODC enzyme activity, we found that the addition of 0.1 mM spermidine to the medium caused a 416% decrease in ODC activity in spe2{Delta} cells but only a 15% decrease in meu1{Delta} cells (Fig. 5A). The double mutant strain had an intermediate phenotype, with a 52% decrease. Differential response to spermidine was also observed at the protein level as shown by immunoblot analysis (Fig. 5B). In the spe2{Delta} strain, the addition of spermidine resulted in undetectable levels of ODC, whereas it had no detectable effect on meu1{Delta} strain. These results show that elevation of ODC levels in meu1{Delta} strains and in spe2{Delta} strains are due to different regulatory systems.



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  		FIG. 5.
ODC activity and protein levels in response to spermidine and MTA. A, yeast strain AS3-2a (meu1{Delta}), YOL052C (spe2{Delta}), and AY32 (meu1{Delta} spe2{Delta}) were grown in synthetic complete polyamine-free medium in the presence or absence of 0.1 mM spermidine for 24 h. Extracts were prepared and analyzed for ODC activity. Error bars indicate S.E. of three measurements. B, same strains as in A analyzed for ODC protein levels by immunoblot. C, yeast strains AS3-2a (meu1{Delta}), AS3-2a (MEU1), and YOL052C (spe2{Delta}) were grown on synthetic complete polyamine-free medium in the presence or absence of 5 mM MTA for 24 h. Extracts were prepared and analyzed as above.

 
MTA Has No Effect on ODC Protein Levels—MTA, the substrate of MTAP, is known to be a strong competitive inhibitor of spermine synthase, S-adenosylhomocysteine hydrolase, and other methyltransferases (38–40). We tested the possibility that elevated MTA levels could cause the elevation of ODC levels we observed in meu1{Delta} yeast. We grew cultures of MEU1, meu1{Delta}, and spe2{Delta} yeast with and without added 5 mM MTA to medium, and examined ODC protein by immunoblot (Fig. 5C). We saw no significant increase in ODC levels of any of the three strains by the addition of exogenous MTA. This result indicates that build-up of MTA is not responsible for the elevated ODC levels observed in meu1{Delta} yeast.

Mutations in E1 and E2 Homologues Affect ODC and Polyamine Levels—Given the result above, we reasoned that a salvage pathway metabolite downstream of MTA might play a key role in repressing ODC levels. To test this hypothesis, we examined ODC levels in yeast strains deleted for genes similar to two known salvage pathway enzymes from Klebsiella oxytoca. The E1 protein from Klebsiella catalyzes the conversion of 2,3-diketo-5-methylthio-1-phosphopentene to 1,2-dihydroxy-3-keto-5-methylthiopentene, whereas the E2 protein catalyzes the subsequent formation of 2-keto-4-methylthiobutyrate (see Fig. 1) (6, 41–43). We searched the S. cerevisiae genome for open reading frames similar to these proteins and found one for each gene. Yeast YEL038W encodes a protein that is 37% identical to Klebsiella E1, whereas yeast YMR009W encodes a protein with 26% identity to E2. We obtained deletions of each of these genes from the Saccharomyces Genome Deletion Project (28). Deletion strains for each of these proteins were viable, and whole cell extracts were prepared and examined for polyamine levels, ODC activity, and protein levels. E1 and E2 deletion strains had elevated ODC activity and protein levels compared with the MEU1 control strain (see Figs. 3B and 4A). However, neither strain had quite the same level of induction as observed in the meu1{Delta} strain. The polyamine profiles also showed an intermediate effect (Fig. 3A). Spermidine levels were clearly elevated in the E1 and E2 mutants, but putrescine was still below the limit of our detection. These results show that mutations in enzymes downstream of MTAP cause elevated ODC levels and elevated levels of spermidine.

MTOB Repression of ODC in meu1{Delta}—Based on the findings above and our knowledge of the biochemical pathway, we reasoned that perhaps MTOB was a negative regulator of ODC. We tested this hypothesis by growing the various mutants in medium in which 1 mM MTOB was substituted for methionine. Yeast grown in MTOB had identical doubling times as yeast grown in methionine (data not shown). However, immunoblot analysis of ODC clearly shows that the addition of MTOB dramatically abolishes the elevated ODC levels observed in meu1, E1, and E2 mutants (Fig. 6). This finding shows that MTOB represses ODC protein levels.



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  		FIG. 6.
Steady-state ODC protein levels in response to MTOB. Yeast strains AS3-2a (MEU1), AS3-2a (meu1{Delta}), YEL038W (E1), and YMR009W (E2{Delta}) were grown in SC medium in the presence or absence of 0.1 mM MTOB for 24 h. Extracts were prepared, and immunoblot analysis of ODC was preformed as described under "Experimental Procedures."

 
MTAP and MTOB Repress ODC in Human Tumor Cells— Previously, we have described the creation of isogenic MTAP-positive and MTAP-negative MCF-7 breast adenocarcinoma cell lines and showed that MTAP caused a 3-fold decrease in total polyamine levels (see Table III in Ref. 1). To see if this repression might be related to ODC repression, we measured ODC activity in two MTAP-expressing and two MTAP-nonexpressing clones (Fig. 7A). Depending on the clones compared, there is a 3–9-fold drop in ODC activity in the MTAP-expressing compared with the nonexpressing cells. These results show that MTAP represses ODC in mammalian cells as well as yeast.



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  		FIG. 7.
ODC activity in mammalian cells. A, MCF-7 cells were stably transfected with either an MTAP-expressing construct (MTAP4 and MTAP8) or a construct that expresses a mutant nonfunctional MTAP (D220A4 and D220A6) (1). Cells were grown to 80% confluence, whole cell extracts were prepared, and ODC activity was measured as described under "Experimental Procedures." Error bars indicate S.E. of three measurements. B, MTAP-deleted MIA PaCa-2 pancreatic adenocarcinoma cells were plated as equal density and grown for 24 h in media containing the indicated levels of methionine or MTOB. Cells were then harvested, extracts were prepared, and ODC activity was measured as described under "Experimental Procedures." Error bars indicate S.E. of three measurements.

 
We also examined whether exposure to MTOB could repress ODC activity in mammalian cells. For these experiments, we used MCF-7 cells and MIAPaca-2 cells, a pancreatic adenocarcinoma cell line deleted for MTAP (31). Cells were grown in regular methionine-containing medium and then placed in either medium with methionine or lacking methionine and supplemented with either 0.2 or 2 mM MTOB. Previously, we have shown that MCF-7 cells can utilize MTOB as a source of fixed sulfur and have identical doubling times on either methionine or MTOB (44). We found similar growth results with MIAPaca-2 cells (data not shown). After 24 h, cells were harvested, and ODC activity was determined (Fig. 7B). Cells from both lines treated with either concentration of MTOB had significant decreases in ODC activity compared with cells growing in 100 µM methionine. These results indicate that MTOB acts as a negative regulator of ODC activity in mammalian cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we demonstrate that the deletion of MTAP causes activation of ODC, resulting in elevated polyamine pools. Our data indicate that a specific downstream salvage pathway metabolite, MTOB, may be a key player in this process. In yeast, mutations in putative salvage pathway genes located between MTAP and MTOB caused elevated ODC protein and activity levels. In both MTAP-deleted yeast and human cells, the addition of MTOB to the medium causes a substantial reduction in ODC activity. Our studies cannot determine whether MTOB is a direct regulator of ODC or whether some metabolite of MTOB is involved. Studies using BAF3 murine lymphoid cells have demonstrated that MTOB can be converted to malondialdehyde and methional in cell extracts (45). Interestingly, MTOB and methional at high concentrations can induce apoptosis in BAF3 cells. It has been shown that drugs that cause depletion of polyamines can sometimes also cause apoptosis in tumor-derived cell lines (46). Thus, it is possible that in BAF3 cells, MTOB may cause apoptosis by depletion of polyamine pools.

We found in S. cerevisiae that mutations in the homologues from Klebsiella E1 and E2 had more modest effects on ODC activation than MTAP deletion. If MTOB were the only inhibitor of ODC, one would expect levels to be identical. There are two possible explanations. First, it may be that there are alternate enzymatic pathways in yeast that can convert methylthioribose-1-phosphate to MTOB, and thus the E1 and E2 mutants may have some low level of MTOB present. Consistent with this idea, Dibner et al. (47) reported that methylthioribose-1-phosphate could be converted to 2-hydroxy-4-methylthiobutanoic acid, which could then be converted to MTOB. Alternatively, other metabolites in the pathway may also inhibit ODC levels. For example, MTOB and methylthioribose 1-phosphate may both be repressors of ODC.

In this paper, we show several lines of evidence that the yeast MEU1 gene encodes MTAP, including amino acid similarity, requirement of MEU1 for enzyme activity, and growth on MTA-containing medium. The MEU1 gene was initially identified in a genetic screen designed to isolate mutations affecting transcriptional regulation of ADH2 (26). Why did MTAP come up in such a screen? Our finding that MTAP deletion alters polyamine homeostasis by overexpression of ODC suggests a possible explanation. Recent evidence indicates that there is a significant link between polyamines, enzymes involved in chromatin remodeling, and transcriptional regulation. In yeast, it has been found that mutations that reduce polyamine levels are able to bypass the need for the core histone deacetylase, GCN5, for the expression of certain genes (48). In ODC overexpressing transgenic mice, both intrinsic histone acetyltransferase and deacetylase activities are elevated (49, 50). Alterations in histone acetyltransferase and histone deacetylase activities would be expected to have effects on a large number of genes (51). Consistent with this hypothesis, microarray analysis of 15,000 expressed sequence tags in isogenic MTAP+ and MTAP– MCF-7 cells indicates that over 200 genes are either 2-fold induced or repressed by MTAP expression.2

ODC levels in yeast and mammalian cells are regulated post-transcriptionally. In higher cells, the antizyme protein targets ODC for degradation by the proteosome (52). Antizyme production requires a frame-shifting event that is greatly stimulated when polyamines are absent. S. cerevisiae lacks a canonical antizyme but appears to have a similar post-transcriptional mechanism for ODC regulation in response to polyamine depletion (36). Our work here shows that the salvage pathway products also negatively regulate ODC. Thus, ODC is feedback-regulated by both of its downstream pathways. We did not observe a "hyper" increase in the ODC levels in the double mutant, suggesting that both feedback processes may be affecting the same underlying mechanism (i.e. disruption of either feedback loop results in elevated ODC).

Our findings also support our earlier hypothesis that MTAP acts as a tumor suppressor by altering polyamine pools. Previously, our laboratory has shown that expression of MTAP in MTAP-deleted MCF-7 cells suppresses anchorage-independent growth and tumor formation in SCID mice and causes significant reduction in polyamine levels (1). This reduction in polyamines is easily explained by inhibition of ODC by MTAP. We also demonstrated in our previous work that the addition of putrescine could partially restore anchorage-independent growth to MTAP-expressing cells, indicating that at least part of the ability of MTAP to suppress tumorigenicity is due to its affect on polyamine production. However, it is possible that MTAP deletion in tumor cells may be affecting other pathways in addition to polyamine biosynthesis that contribute to tumorigenicity. Future experiments will focus on answering this question.


   FOOTNOTES
 
* This work was supported by United States Army Grant DAMD1797-1-7707, United States Public Health Service Grant CA-22153, National Institutes of Health Core Grant CA-06927, and an appropriation from the Commonwealth of Pennsylvania. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111-2497. Tel.: 215-728-3030; Fax: 215-214-1623; E-mail: wd_kruger{at}fccc.edu.

1 The abbreviations used are: MTAP, methylthioadenosine phosphorylase; MTA, methylthioadenosine; ODC, ornithine decarboxylase; MTOB, 4-methylthio-2-oxobutanoic acid; HPLC, high pressure liquid chromatography. Back

2 B. Tang and W. D. Kruger, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Martin Hoyt and Phil Cofino for the use of the yeast ODC antiserum. We also thank Randy Strich, Elizabeth Henske, and Eric Moss for critical reading of the manuscript. We also acknowledge the work of the Sequencing and Cell Culture Facilities at Fox Chase Cancer Center.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639–6644[Abstract/Free Full Text]
  2. Della Ragione, F., Russo, G., Oliva, A., Mastropietro, S., Mancini, A., Borrelli, A., Casero, R. A., Iolascon, A., and Zappia, V. (1995) Oncogene 10, 827–833[Medline] [Order article via Infotrieve]
  3. Batova, A., Diccianni, M. B., Nobori, T., Vu, T., Yu, J., Bridgeman, L., and Yu, A. L. (1996) Blood 88, 3083–3090[Abstract/Free Full Text]
  4. Nobori, T., Takabayashi, K., Tran, P., Orvis, L., Batova, A., Yu, A. L., and Carson, D. A. (1996) Proc. Natl. Acad. Sci. U. S. A 93, 6203–6208[Abstract/Free Full Text]
  5. Garcia-Castellano, J. M., Villanueva, A., Healey, J. H., Sowers, R., CordonCardo, C., Huvos, A., Bertino, J. R., Meyers, P., and Gorlick, R. (2002) Clin. Cancer Res. 8, 782–787[Abstract/Free Full Text]
  6. Wray, J. W., and Abeles, R. H. (1995) J. Biol. Chem. 270, 3147–3153[Abstract/Free Full Text]
  7. Auvinen, M., Paasinen, A., Andersson, L. C., and Holtta, E. (1992) Nature 360, 355–358[CrossRef][Medline] [Order article via Infotrieve]
  8. Pegg, A. E. (1988) Cancer Res. 48, 759–774[Abstract/Free Full Text]
  9. O'Brien, T. G., Megosh, L. C., Gilliard, G., and Soler, A. P. (1997) Cancer Res. 57, 2630–2637[Abstract/Free Full Text]
  10. Toohey, J. I. (1978) Biochem. Biophys. Res. Commun. 83, 27–35[CrossRef][Medline] [Order article via Infotrieve]
  11. Trackman, P. C., and Abeles, R. H. (1981) Biochem. Biophys. Res. Commun. 103, 1238–1244[CrossRef][Medline] [Order article via Infotrieve]
  12. Trackman, P. C., and Abeles, R. H. (1983) J. Biol. Chem. 258, 6717–6720[Abstract/Free Full Text]
  13. Furfine, E. S., and Abeles, R. H. (1988) J. Biol. Chem. 263, 9598–9606[Abstract/Free Full Text]
  14. Myers, R. W., and Abeles, R. H. (1990) J. Biol. Chem. 265, 16913–16921[Abstract/Free Full Text]
  15. Backlund, P. S., Jr., and Smith, R. A. (1981) J. Biol. Chem. 256, 1533–1535[Abstract/Free Full Text]
  16. Backlund, P. S., Jr., Chang, C. P., and Smith, R. A. (1982) J. Biol. Chem. 257, 4196–4202[Abstract/Free Full Text]
  17. Backlund, P. S., Jr., and Smith, R. A. (1982) Biochem. Biophys. Res. Commun. 108, 687–695[Medline] [Order article via Infotrieve]
  18. Cone, M. C., Marchitto, K., Zehfus, B., and Ferro, A. J. (1982) J. Bacteriol. 151, 510–515[Abstract/Free Full Text]
  19. Marchitto, K. S., and Ferro, A. J. (1985) J. Gen. Microbiol. 131, 2153–2164[Medline] [Order article via Infotrieve]
  20. Schmid, M., Malicki, D., Nobori, T., Rosenbach, M. D., Campbell, K., Carson, D. A., and Carrera, C. J. (1998) Oncogene 17, 2669–2675[CrossRef][Medline] [Order article via Infotrieve]
  21. Stadler, W. M., and Olopade, O. I. (1996) Urol. Res. 24, 239–244[CrossRef][Medline] [Order article via Infotrieve]
  22. Nobori, T., Karras, J. G., Della Ragione, F., Waltz, T. A., Chen, P. P., and Carson, D. A. (1991) Cancer Res. 51, 3193–3197[Abstract/Free Full Text]
  23. Cohen, S. S. (1998) in A Guide to the Polyamines, pp. 296–319, Oxford University Press, Oxford
  24. Megosh, L., Gilmour, S. K., Rosson, D., Soler, A. P., Blessing, M., Sawicki, J. A., and O'Brien, T. G. (1995) Cancer Res. 55, 4205–4209[Abstract/Free Full Text]
  25. Thomas, D., Becker, A., and Surdin-Kerjan, Y. (2000) J. Biol. Chem. 275, 40718–40724[Abstract/Free Full Text]
  26. Donoviel, M. S., and Young, E. T. (1996) Genetics 143, 1137–1148[Abstract]
  27. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19–27[Abstract/Free Full Text]
  28. Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., Andre, B., Arkin, A. P., Astromoff, A., El-Bakkoury, M., Bangham, R., Benito, R., Brachat, S., Campanaro, S., Curtiss, M., Davis, K., Deutschbauer, A., Entian, K. D., Flaherty, P., Foury, F., Garfinkel, D. J., Gerstein, M., Gotte, D., Guldener, U., Hegemann, J. H., Hempel, S., Herman, Z., Jaramillo, D. F., Kelly, D. E., Kelly, S. L., Kotter, P., LaBonte, D., Lamb, D. C., Lan, N., Liang, H., Liao, H., Liu, L., Luo, C., Lussier, M., Mao, R., Menard, P., Ooi, S. L., Revuelta, J. L., Roberts, C. J., Rose, M., Ross-Macdonald, P., Scherens, B., Schimmack, G., Shafer, B., Shoemaker, D. D., Sookhai-Mahadeo, S., Storms, R. K., Strathern, J. N., Valle, G., Voet, M., Volckaert, G., Wang, C. Y., Ward, T. R., Wilhelmy, J., Winzeler, E. A., Yang, Y., Yen, G., Youngman, E., Yu, K., Bussey, H., Boeke, J. D., Snyder, M., Philippsen, P., Davis, R. W., and Johnston, M. (2002) Nature 418, 387–391[CrossRef][Medline] [Order article via Infotrieve]
  29. Sherman, F. (1991) Methods Enzymol. 194, 3–21[CrossRef][Medline] [Order article via Infotrieve]
  30. Balasundaram, D., Tabor, C. W., and Tabor, H. (1991) Proc. Natl. Acad. Sci. U. S. A 88, 5872–5876[Abstract/Free Full Text]
  31. Chen, Z. H., Olopade, O. I., and Savarese, T. M. (1997) Mol. Pharmacol. 52, 903–911[Abstract/Free Full Text]
  32. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  33. Tyagi, A. K., Tabor, C. W., and Tabor, H. (1983) Methods Enzymol. 94, 135–139[Medline] [Order article via Infotrieve]
  34. Cohn, M. S., Tabor, C. W., and Tabor, H. (1983) Methods Enzymol. 94, 231–234[Medline] [Order article via Infotrieve]
  35. Vujcic, S., Halmekyto, M., Diegelman, P., Gan, G., Kramer, D. L., Janne, J., and Porter, C. W. (2000) J. Biol. Chem. 275, 38319–38328[Abstract/Free Full Text]
  36. Gupta, R., Hamasaki-Katagiri, N., White Tabor, C., and Tabor, H. (2001) Proc. Natl. Acad. Sci. U. S. A 98, 10620–10623[Abstract/Free Full Text]
  37. Toth, C., and Coffino, P. (1999) J. Biol. Chem. 274, 25921–25926[Abstract/Free Full Text]
  38. Williams-Ashman, H. G., Seidenfeld, J., and Galletti, P. (1982) Biochem. Pharmacol. 31, 277–288[CrossRef][Medline] [Order article via Infotrieve]
  39. Pegg, A. E., Borchardt, R. T., and Coward, J. K. (1981) Biochem. J. 194, 79–89[Medline] [Order article via Infotrieve]
  40. Ferro, A. J., Vandenbark, A. A., and MacDonald, M. R. (1981) Biochem. Biophys. Res. Commun. 100, 523–531[CrossRef][Medline] [Order article via Infotrieve]
  41. Myers, R. W., Wray, J. W., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785–24791[Abstract/Free Full Text]
  42. Balakrishnan, R., Frohlich, M., Rahaim, P. T., Backman, K., and Yocum, R. R. (1993) J. Biol. Chem. 268, 24792–24795[Abstract/Free Full Text]
  43. Dai, Y., Wensink, P. C., and Abeles, R. H. (1999) J. Biol. Chem. 274, 1193–1195[Abstract/Free Full Text]
  44. Tang, B., Li, Y. N., and Kruger, W. D. (2000) Cancer Res. 60, 5543–5547[Abstract/Free Full Text]
  45. Quash, G., Roch, A. M., Chantepie, J., Michal, Y., Fournet, G., and Dumontet, C. (1995) Biochem. J. 305, 1017–1025[Medline] [Order article via Infotrieve]
  46. Schipper, R. G., Penning, L. C., and Verhofstad, A. A. (2000) Semin. Cancer Biol. 10, 55–68[CrossRef][Medline] [Order article via Infotrieve]
  47. Dibner, J. J., Durley, R. C., Kostelc, J. G., and Ivey, F. J. (1990) J. Nutr. 120, 553–560[Abstract/Free Full Text]
  48. Pollard, K. J., Samuels, M. L., Crowley, K. A., Hansen, J. C., and Peterson, C. L. (1999) EMBO J. 18, 5622–5633[CrossRef][Medline] [Order article via Infotrieve]
  49. Hobbs, C. A., and Gilmour, S. K. (2000) J. Cell. Biochem. 77, 345–360[CrossRef][Medline] [Order article via Infotrieve]
  50. Hobbs, C. A., Paul, B. A., and Gilmour, S. K. (2002) Cancer Res. 62, 67–74[Abstract/Free Full Text]
  51. Kuo, M. H., and Allis, C. D. (1998) BioEssays 20, 615–626[CrossRef][Medline] [Order article via Infotrieve]
  52. Coffino, P. (2001) Nat. Rev. Mol. Cell. Biol. 2, 188–194[CrossRef][Medline] [Order article via Infotrieve]
  53. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J., Hieter, P., and Boeke, J. D. (1998) Yeast 14, 115–132[CrossRef][Medline] [Order article via Infotrieve]

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