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[Cancer Research 60, 5543-5547, October 1, 2000]
© 2000 American Association for Cancer Research

Molecular Biology and Genetics

Defects in Methylthioadenosine Phosphorylase Are Associated with but not Responsible for Methionine-dependent Tumor Cell Growth1

Baiqing Tang, Yunan N. Li and Warren D. Kruger2

Division of Population Science, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A large proportion of human tumor-derived cell lines and primary tumor cells show methionine-dependent growth. This phenomenon refers to the ability of cells to grow in media containing methionine and the inability of cells to grow in media supplemented with methionine’s precursor, homocysteine (Hcy). Methionine can be formed by two different pathways, the recycling pathway and the salvage pathway. To discover the basis for methionine-dependent growth, we have analyzed 12 tumor cell lines and 2 non-tumor-derived cell lines for defects in two key genes in different methionine synthetic pathways. We found little evidence that defects in methionine synthase expression or mutations in the MS gene are correlated with methionine-dependent growth. However, we did find a correlation between methionine-dependent growth and defects in expression of methylthioadenosine phosphorylase (MTAP), a key enzyme in the salvage pathway. Three of the four cell lines lacking detectable MTAP protein were unable to grow in Hcy-containing media, whereas all six of the MTAP-positive cell lines tested showed strong growth. However, when we introduced MTAP cDNA into MTAP-deficient MCF-7 cells, the resulting cell line was still defective in growth on Hcy, although it could now grow on the salvage pathway precursor methylthioadenosine. These findings indicate that salvage pathway defects are not causally related to methionine-dependent growth.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methionine dependence denotes the inability of cells to grow in vitro when methionine is replaced with Hcy3 in the culture medium (1 , 2) . The phenomenon has been demonstrated in a large number of malignant cell lines and in primary cultures of human malignant tumors (3 , 4) . With few exceptions, nontransformed cells or tissue is Met independent (1 , 5 , 6) . These observations have led to an interest in using recombinant methioninase as an anticancer agent (7) .

The enzyme that converts Hcy to methionine, MS, is a conceivable site of a defect in Met-dependent tumor cells. This enzyme catalyzes a cobalamin-dependent reaction that transfers a methyl group from 5'-methyltetrahydrofolate onto Hcy to form methionine (see Fig. 1Citation ). Although some studies of methionine-dependent cells have shown decreases in MS activity measured in cell extracts (8 , 9) , other studies have not (10 , 11) . Additional studies have presented evidence that some methionine-dependent cells have defects in cobalamin (B12) metabolism (9 , 12) . Thus, the role of MS in methionine dependence is still unclear.



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  		Fig. 1. Methionine biosynthetic pathways. The recycling and salvage pathways are presented. Relevant enzymes are shown in bold.

 
Other literature has emphasized the importance of the methionine salvage pathway in explaining methionine-dependent growth (13) . This pathway salvages the MTA that is released after S-adenosylmethionine donates an aminopropyl group for polyamine biosynthesis. The MTA is then converted to adenine and methionine in a multistep enzymatic procedure (Fig. 1)Citation . Ogier et al. (14) found that four different methionine-dependent cell lines could be rescued by addition of the penultimate compound in the salvage pathway, MTOB. It has been claimed that Met-dependent tumor cells do not lack the ability to form methionine from Hcy but rather have a metabolic defect in the salvage pathway (13) . Indeed, one of the key enzymes in this pathway, MTAP, is frequently absent in many kinds of cancer cells and primary tumors including leukemias, brain tumors, non-small cell lung cancers, and bladder cancers (15, 16, 17, 18, 19) .

To understand the mechanisms of methionine-dependent growth in tumor cells, we surveyed 12 human tumor cell lines for alterations in the MS and MTAP gene and reexamined the growth behavior of a subset of these cell lines in media containing various methionine precursors. Our results indicate that although methionine-dependent growth and MTAP deletion frequently occur together in the same cells, MTAP deletion is not responsible for the methionine-dependent growth phenotype.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
L-Met, Hcy, and MTA were purchased from Sigma Chemical Co. (St. Louis, MO). MTOB was obtained from Aldrich Chemical Co. (Milwaukee, WI).

Cell Lines and Media.
All of the cell lines used in this study are listed in Table 1Citation . FC1010 cells were isolated in the FCCC tissue culture facility. All other tumor cell lines were purchased from ATCC. Cell lines from ATCC were grown in the media recommended by ATCC. FC1010 was grown in RPM 1640 supplemented with dialyzed 10% FBS. The corresponding type of Met-free media supplemented with 10 µM B12 were used to test Met dependence. Eagle’s Met-free MEM was ordered from Sigma, and Met-free DMEM was obtained from Life Technologies, Inc. (Grand Island, NY).


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  		Table 1 Cell lines used in this study

 
Cell Growth Assays.
Cells (1–5 x 105) were plated depending on the type of cell line and the size of the cells. Cells were seeded into 3-ml of Met-free medium plus FBS in 6-well plates or in 10 ml of medium in T25 flasks. Four h later, 100 µM of either Hcy, MTA, MTOB, or Met was added. Each treatment was carried out in duplicate or triplicate at the same time. After 4 days, the cells were either trypsinized or scraped off the dishes using cell scrapers. Cell growth was assessed by cell counting and/or measuring the total proteins using the Bio-Rad protein assay dye kit. Total protein was found to correlate very closely with cell counting (data not shown). Each value is the average of two or three repeats.

Isolation of Total RNA, RT-PCR, and Sequencing.
Total RNA was isolated from cultured cells (1–5 x 106) using Perfect RNA Total RNA Isolation Kit (5'-3', Inc., Boulder, CO). First-strand cDNA synthesis was performed on 1 µg of total RNA from an individual cell line using oligo(dT)16 as a primer with the Advantage RT-for-PCR kit (Clonetech, Palo Alto, CA). PCR was carried out using Advantage KlenTaq Polymerase Mix (Clonetech). PCR-DNAs were either sequenced directly or cloned into pCR2.1 vector (Invitrogen, San Diego, CA), and several individual clones were pooled for sequencing.

SDS-PAGE and Western Blotting.
Cells were lysed and sonicated in 1x PBS containing 1 mM phenylmethylsulfonyl fluoride (Sigma). The lysate was suspended in 1x SDS sample buffer, and proteins were separated by SDS-PAGE. Proteins were then transferred onto an Immobilon P membrane (Millipore Corp., Bedford, MA). The subsequent steps of Western blotting were performed using the Immun-Lite Assay Kit from Bio-Rad according to the manufacturer’s instructions. The primary antibodies used in this study were MS anti-sera [from Dr. Ruma Banerjee’s laboratory (University of Nebraska, Omaha, NE; Ref. 20 )] and MTAP antibody produced from chicken yolk (from Dr. Dennis Carson’s laboratory, University of San Diego, CA).

Cloning and Transfection of MTAP.
MTAP cDNAs were obtained by PCR using Raji cell cDNA as template with primers 5'-AATTCCGCTCCGCACTGCTCACTCCCG and 5'-CTCCTGGGCAGCCATGCTACTTTAATG. MTAP cDNA was cloned into the eukaryotic expression vector pCR3.1 (Invitrogen) and then transformed into TOP10F competent cells according to the manufacturer’s instructions. The orientation of the insert was determined by digesting plasmid DNA with HincII, and the clones were sequenced to screen for PCR-induced errors. The sense construct was designated pCR:sMTAP, whereas the antisense construct was designated pCR:asMTAP. Five µg of pCR:sMTAP and pCR:asMTAP were used to transfect MCF-7 cells with pCR3.1/Cat expression vector as a positive control by using the Qiagen Superfect Transfection Reagent Kit (Qiagen, Inc., Chatsworth, CA). Twenty-four h after transfection, medium was replaced by MEM with 2 mM L-glutamine, 1.5 mg/ml sodium bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 0.01 mg/ml bovine insulin, 10% FBS, and 600 mg/ml G418 (Life Technologies, Inc.). After culture for 5 weeks in the presence of G418, colonies were picked and subcloned. Six clones were tested for the expression of MTAP protein by Western blot analysis.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
MS Protein Expression and Mutation Analysis.
We examined 12 human tumor and 2 nontransformed cell lines for alterations in MS protein levels (Table 1)Citation . Ten of the cell lines had previously been characterized as having either absolute or partial methionine-dependent growth. We examined MS protein levels by Western analysis. The antiserum was generated against purified porcine MS, but it cross-reacts with human, mouse, and Escherichia coli MS due to the high degree of evolutionary conservation of the MS protein (21) . Among the 14 cell lines, we saw no significant difference in steady-state MS levels, with one exception (Fig. 2)Citation . We observed a slight decrease in MS levels in SK-CO-1, a colon carcinoma-derived cell line that has previously been reported to exhibit partial methionine-dependent growth behavior.



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  		Fig. 2. Western analysis of MS protein. Total cellular protein was separated on 10% SDS-polyacrylamide gel and transferred to nylon membranes as described in "Materials and Methods." p4B6.3 is an E. coli strain overexpressing the METH protein, which is the bacterial homologue of MS.

 
We next examined whether any of the cell lines contained mutations in the MS coding region. RNA from each of the human cell lines was isolated, and RT-PCR was used to amplify the entire MS coding region. The resulting PCR product was then sequenced directly. We did not observe any nonsense or missense mutations in any of the cell lines. However, we did identify several silent changes (Table 2)Citation present in some cell lines, indicating that our methods were sensitive enough to detect heterozygous alterations. These results show that mutations that inactivate the MS protein are not found frequently in methionine-dependent cells.


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  		Table 2 Polymorphisms present in the MS gene

 
MTAP Expression in Tumor Cell Lines.
Next we examined the MTAP status in these 12 tumor cell lines by RT-PCR and Western blotting. The primers used for RT-PCR of MTAP cDNA were derived from the immediate flanking sequences of MTAP open reading frame (22) . We were unable to detect MTAP RNA in 4 of the 12 tumor-derived cell lines (see Fig. 3ACitation ). As a control for the quality of the RNA and the cDNA synthesis, the glyceraldehyde-3-phosphate dehydrogenase gene was amplified successfully in all samples (Fig. 3C)Citation .



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  		Fig. 3. MTAP analysis of tumor-derived cell lines. A, RT-PCR analysis of MTAP expression in the indicated cell lines; B, Western analysis of MTAP in the same cells; C, RT-PCR of glyceraldehyde-3-phosphate dehydrogenase.

 
To confirm the RT-PCR data, we also examined MTAP protein by Western blot. All of the cell lines lacking MTAP RNA as judged by RT-PCR also lacked detectable MTAP protein by Western analysis (Fig. 3B)Citation .

Cell Growth.
We wanted to assess whether failure to express MTAP correlates with a distinguishing growth pattern in media containing methionine and various precursors. We examined the growth of 10 cell lines: (a) 4 MTAP+ tumor cell lines; (b) 4 MTAP- lines; and (c) two nontransformed cell lines (Table 3)Citation . Cell growth was evaluated in the presence of 100 µM methionine, MTA, MTOB, or Hcy (see Fig. 1Citation ). The two nontransformed control cell lines (MRC-5 and FC1010) both showed excellent growth in media containing methionine or MTOB and significant growth in media containing Hcy or MTA. All four of the MTAP+ cell lines tested (HeLa, SK-N-SH, J82, and Raji) exhibited growth behavior that was essentially identical to that of the nontransformed cell lines. This was somewhat surprising because three of these four cell lines (HeLa, SK-N-SH, and J82) have previously been characterized as either absolutely or partially methionine dependent (5) . However, the four MTAP- cell lines (MCF-7, HOS, HT1080, and A172) exhibited very different growth characteristics. As expected, all four MTAP- cell lines were unable to grow in media containing MTA as the sole source of methionine. Interestingly, three of the four MTAP- cell lines were also unable to grow in Hcy-containing media. The single exception was the A172 glioblastoma cell line, which showed no growth in MTA but showed clear growth in Hcy-containing media. Thus, all three of the cell lines that showed absolutely no growth in Hcy-containing media were MTAP-.


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  		Table 3 Cell growth studiesa

 
Reintroduction of MTAP into MCF-7 Cells.
We next tested directly whether deletion of MTAP was responsible for the methionine-dependent growth. Wild-type MTAP cDNA was cloned into expression vector pCR3.1 into the sense (pCR:sMTAP) and antisense (pCR:asMTAP) orientations. These constructs, along with pCR3.1 itself, were used to transfect the MTAP- MCF-7 cells to create stable transfected lines. Western analysis indicated that one of the cell lines transfected with the sense orientation construct, pCR3.1:sMTAP, expressed MTAP levels comparable to Raji cells, a methionine-independent cell line (see Fig. 4Citation ). Control transfectants containing either pCR3.1:asMTAP or pCR3.1 had no detectable MTAP protein.



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  		Fig. 4. MTAP protein in MCF transfectants. Western blots were probed with anti-MTAP antiserum. Lane 1, Raji cell extract. Lane 2, MCF-7 cells transfected with pCR3.1 with MTAP in the sense orientation. Lane 3, MCF-7 cells transfected with pCR3.1 with MTAP in the antisense orientation. Lane 4, MCF-7 cells transfected with pCR3.1 with no insert.

 
We next examined the ability of these cell lines to grow on media containing various sources of sulfur. Cells expressing MTAP were able to grow on media with MTA as the sole source of methionine precursor (see Table 4Citation ), but the control cells were not able to do so. However, no significant improvement in growth on Hcy-containing media was observed. Examination of growth in comparison to Raji cells at 3, 5, and 7 days indicated that no significant growth took place on Hcy-containing media (Table 5)Citation . Thus, expression of MTAP in MCF-7 cells corrected the MTAP- phenotype, but not the methionine-dependent growth phenotype.


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  		Table 4 Growth behavior of MTAP transfected MCF-7 cellsa

 

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  		Table 5 Growth behavior of MTAP-transfected MCF-7 cells compared with Raji cellsa

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results reported here support the view that defects in the MS gene are unlikely to be a major cause of methionine-dependent growth. We failed to identify a single missense or nonsense mutation in the MS gene in 12 tumor cell lines, 10 of which have been previously characterized as being at least partially methionine dependent for growth. Only one cell line, a colon carcinoma-derived line (SK-CO-1), had lower levels of MS protein compared with all of the other cell lines. Interestingly, this cell line was previously classified as only partially methionine dependent.

Although defects in MTAP expression are often observed in methionine-dependent cell lines, these do not appear to be the cause of methionine-dependent growth. Four of the 12 cell lines examined lacked MTAP RNA and MTAP protein. All four of these cell lines could not use MTA as a sulfur source, and three of these cell lines were also unable to use Hcy. These results suggested that MTAP deficiency could be related to methionine-dependent growth. However, when we reintroduced MTAP into one of these cell lines (MCF-7), we corrected the MTA growth defect but not the methionine-dependent growth. Thus, the defect in MTAP expression is separate from methionine-dependent growth.

The gene for MTAP resides on chromosome 9p21 in close proximity to the tumor suppressor gene p16 and is frequently found to be deleted in a variety of cancers (15, 16, 17, 18, 19) . It is generally assumed that MTAP is deleted "by accident" simply because of its proximity to p16. The p16 gene is unusual among tumor suppressor genes in that the mechanism of inactivation generally involves homozygous deletion as opposed to loss of heterozygosity and point mutation (23) . Whether deletion of MTAP is coincidental or gives the tumor cell a selective advantage is unknown. Interestingly, it has recently been described that in a set of non-small cell lung carcinomas, MTAP deletion occurs frequently in the absence of p16 deletion, suggesting that MTAP dysfunction may give the tumor cell some advantage (24) .

One unexpected finding of this study is that many of the cell lines that had previously been characterized as either fully or partially methionine dependent exhibited methionine-independent growth in our hands. For example, two of the cell lines characterized in other studies (5) as showing no growth on Hcy (SK-N-SH and J82) exhibited only a slightly retarded growth on Hcy compared with the two control cell lines. In addition, two other cell lines previously characterized as only slightly independent (HeLa and A172) exhibited growth in Hcy that was essentially undistinguishable from that of our control cells. A possible explanation for these discrepancies is that the cell lines might not be identical to the ones used in the earlier studies. Fiskerstrand et al. (12) have observed that a human glioma cell line that was initially methionine independent became methionine dependent when the passage number increased. Perhaps our cells (most of which were obtained from ATCC) are at earlier passages than the cells used in the study of Mecham et al. (5) .

We observed that although MTAP deletion did not explain methionine-dependent growth, MTAP deletion and methionine-dependent growth frequently occurred together. Three of the four MTAP-deficient cell lines showed no growth on Hcy. The observation that these two phenotypes frequently occur together may be potentially useful in cancer treatment. Cells deleted for MTAP are known to be more sensitive to the effects of purine biosynthetic inhibitors such as methotrexate, L-alonosine, 5,10-dideazatetrahyhydrofolate, and azaserine in vitro (25, 26, 27) . Methionine-dependent tumor cells have been shown to be sensitive to depletion of plasma methionine in vivo. Methionine-dependent human cancer xenografts in nude mice grow much more slowly when the mice are put on a methionine-free diet (28) . In addition, methionine-dependent cancer xenografts can have their growth reduced by the use of recombinant methioninase to lower plasma methionine levels (29) . Thus, by combining purine inhibitors with plasma methionine-lowering procedures, it may be possible to get increased selective killing of MTAP- methionine-dependent tumors.

Our observations still leave the question of what is the molecular basis of methionine dependency unanswered. One possibility is that methionine-dependent cells could have defects in MTHFR. This enzyme makes methyltetrahydrofolate, which is the methyl donor for the conversion of Hcy to methionine. It has been reported that in some ovarian carcinomas, there is loss of heterozygosity at the MTHFR locus that is associated with decreased activity (30) . However, RT-PCR analysis indicates that none of the cell lines used in this study have defects in MTHFR expression (data not shown). Another possible mechanism for methionine-dependent growth involves impairment in the uptake/metabolism of cobalamin. Cobalamin is a vitamin cofactor required for MS function. Studies of the methionine dependence of P60 human glioma cells indicate that these cells have reduced levels of both methylcobalamin and adenosylcobalamin (12) . Methionine-dependent MeWoLC1 melanoma-derived cells also seem to have defects in cobalamin metabolism (9) . Complementation analysis suggests that the defect is in the same gene as that observed in cells from individuals suffering from the cblC inborn error of metabolism (31) . However, this same group also showed that inactivation of the cblC gene is unlikely to be involved in the methionine dependence of most other methionine-dependent tumors, including the MCF-7 cells studied here.

A final possibility is that methionine dependence results not from a defect in the production of methionine but rather from overutilization of methionine. Several studies have demonstrated that methionine-dependent cells tend to have reduced ratios of S-adenosylmethionine:S-adenosylhomocysteine, suggesting that these cells have increased rates of transmethylation (32, 33, 34) . Thus, it may be that the production of methionine simply cannot keep up with its utilization.

In summary, the studies described here suggest that mutation in MS or MTAP is not the cause of the methionine-dependent growth observed in most tumor cells. However, MTAP deletion and methionine dependence often occur in the same cells, suggesting that a combination of purine inhibitors and methionine starvation may be a reasonable approach to kill these cells. Future efforts toward understanding the genetic basis for methionine dependence could help development of a genetic test to identify methionine-dependent tumors, and this information could be incorporated into studies examining the efficacy of methionine-lowering treatments.


   ACKNOWLEDGMENTS
 
We thank Dr. Dennis Carson for the use of MTAP antiserum and Dr. Ruma Banerjee for the MS antiserum. We also thank A. Bellacosa, R. Raftogianis, and E. Moss for helpful comments and critical reading of this manuscript.


   FOOTNOTES
 
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.

1 Supported in part by United States Army Grant DAMD17-97-1-7707, a grant from the Bugher Foundation, USPHS Grant CA06927 from the NIH, and by an appropriation from the Commonwealth of Pennsylvania. Back

2 To whom requests for reprints should be addressed, at Division of Population Science, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Phone: (215) 728-3030; E-mail: wd_kruger{at}fccc.edu Back

3 The abbreviations used are: Hcy, homocysteine; MS, methionine synthase; MTA, methylthioadenosine; MTOB, 4-methylthio-2-oxobutanoate; MTAP, methylthioadenosine phosphorylase; ATCC, American Type Culture Collection; FBS, fetal bovine serum; RT-PCR, reverse transcription-PCR; MTHFR, methylenetetrahydrofolate reductase. Back

Received 4/ 7/00. Accepted 8/ 2/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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