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[Cancer Research 59, 4194-4199, September 1, 1999]
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[Cancer Research 59, 4194-4199, September 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Metallothionein Is Overexpressed by Hamster Fibroblasts Selected for Growth in 15 pM Folinic Acid and Provides a Growth Advantage in Low Folate1

Wei-Yong Zhu and Peter W. Melera2

Department of Biochemistry and Molecular Biology [W. Z., P. W. M.] and the Greenebaum Cancer Center [P. W. M.], University of Maryland School of Medicine, Baltimore, Maryland 21201


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
DC-3F/FA3 (FA3) cells, selected for growth in folic acid-free medium containing dialyzed serum and 15 pM [6S] -folinic acid, and parental DC-3F cells were compared by mRNA differential display to identify genetic changes occurring during selection. One of the genes found to be overexpressed in FA3 cells was metallothionein II (MT-II). Northern blots using a full-length hamster MT-II cDNA probe that recognizes both MT-I and MT-II RNA showed that the steady-state level of MT mRNA was elevated at least 10-fold in FA3 cells and in two other selected clones, FA7 and FA14, as well. Southern blot analysis of HindIII-digested genomic DNA indicated that amplification of neither the MT-I nor MT-II gene had occurred, and measurements of MT mRNA decay rates in the presence of actinomycin D suggested that no changes in its half-life had taken place. Hence, overexpression was due to an increase in transcription from the normal gene complement. In FA3 cells, the MT mRNA expression level was found to be directly sensitive and inversely proportional to media folate concentrations, whereas in DC-3F cells it was not, suggesting that MT gene expression is differentially regulated in these two cell lines. Overexpression of MT-II in transfected DC-3F cells was unable to support growth in 15 pM folinic acid. However, when plated in 15 nM folinic acid, a growth rate similar to FA3 cells was observed, whereas sham-transfected controls and double transfectants expressing antisense MT-II RNA and control levels of MT-II protein ceased to grow. Hence, overexpression of MT-II provides a growth advantage in low folate.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The folate vitamins serve as cofactors for a number of biochemical reactions that are pivotal for the maintenance of metabolic stasis. Mammalian cells cannot carry out the de novo synthesis of folate, and although the folate cycle is responsible for maintaining intracellular folate homeostasis, cells must rely on uptake of serum folate to maintain pool equilibrium and ensure survival. Several studies have shown that transfection of FR{alpha}3 cDNA into cells that do not constitutively express the receptor permits greater proliferation and survival compared with controls when cultured in low extracellular folate (1) . It has also been shown that cells selected for growth in low folate up-regulate FR{alpha} expression (1) . Less is known, however, about other genetic alterations or metabolic changes that emerge during selection for growth under conditions of severe folate depletion. To address this question, we isolated clones from the Chinese hamster lung cell line DC-3F that had been cultured in folic acid-free medium with dialyzed fetal bovine calf serum and in which the sole folate source was 15 pM [6S]-folinic acid (2) . To identify genetic changes emerging during this rigorous selection, mRNA differential display was used to compare a selected clone, DC-3F/FA3 (FA3), with its parent DC-3F.

MT genes encode low molecular weight, cysteine-rich proteins that bind heavy metals (3) . Within the MT gene family, MT-I and MT-II, are known to be involved in heavy metal homeostasis and detoxification. Numerous studies have shown that MT gene transcription is induced by metals via multiple metal-responsive elements and by glucocorticoid response elements present in the 5'-regulatory regions of the gene (3 , 4) . Furthermore, other stimuli such as IFN, lipopolysaccharide, and UV-irradiation also elevate MT gene expression, suggesting that the gene product serves multiple cellular functions in addition to metal detoxification (5) . In this study, we show that folate restriction leads to an overexpression of MT-II and that it is sufficient to provide a growth advantage to cells in low folate media.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Materials.
All tissue culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). [6S]-Folinic acid was obtained from SAPEC, S.A. Fine Chemicals (Barbengo, Switzerland).

Cell Culture.
Parental Chinese hamster lung fibroblast cells (DC-3F) were maintained in MEM with Ham’s F12 nutrient mixture (MEM/F12), supplemented with 5% heat-inactivated fetal bovine serum (HyClone, Logan, UT), and 1% penicillin/streptomycin solution. Clones selected for growth under conditions of severe folate restriction, FA3, FA7, and FA14, have been described elsewhere.4 The clones were maintained in MEM/F12 without hypoxanthine, thymidine, or folic acid and were supplemented with 5% dialyzed, heat-inactivated fetal bovine serum and 15 pM [6S]-folinic acid (FA medium).

mRNA Differential Display.
Differential display was performed using RNA from FA3 and DC-3F cells according to the protocol supplied with an RNAimage kit (GenHunter Corp., Nashville, TN). Briefly, 0.2 µg of DNase I (GenHunter Corp.)-treated total RNA was reverse transcribed with H-T11M primers (where M may be G, A, or C), followed by PCR amplification in the presence of [{alpha}-33P]dATP (NEN) using the corresponding H-T11M primer, downstream, and one of the arbitrary primers supplied with the kit, AP1-AP40, upstream. The PCR-amplified fragments were separated on a 6% denaturing polyacrylamide gel. The gel was dried and exposed to Kodak XAR film, and cDNA representing differentially expressed mRNAs was excised from the dried gels and reamplified by PCR for 40 cycles using the corresponding set of the primers. The reamplified cDNA fragments were cloned into PCR-TRAP vectors (GenHunter) and used as probes in Northern blots to verify their differential expression in FA3 and DC-3F cells.

Northern Blot Analysis.
Twenty µg of total RNA isolated with TRIzol LS reagent (Life Technologies, Inc.) were fractionated on a 1% agarose gel and blotted overnight onto GeneScreen Plus nylon membranes. These were prehybridized for 4 h at 42°C in 5x SSPE, 5x Denhardt’s solution, 1% SDS, 50% formamide, 10% dextran sulfate, and 100 µg/ml denatured and fragmented salmon sperm DNA. cDNA probes labeled with [{alpha}-32P]dCTP using the Random Primer Labeling System (Life Technologies, Inc.) were denatured and added to the prehybridization solution. Hybridization was allowed to proceed for 24 h at 42°C. After washing, the blots were analyzed by autoradiography using Kodak XAR film. As a control for variations in RNA loading, the membranes were then stripped and rehybridized with a labeled ß-actin cDNA probe.

mRNA Half-life Determination.
Cells were plated in the appropriate medium, and when growth reached 70–80% confluence, actinomycin D was added to a final concentration of 5 µg/ml (6) . At various time points (0, 3, 6, 8, and 24 h), total RNA was then extracted as described above and the levels of MT-II mRNA were determined by Northern analysis. After being stripped, the blots were reprobed with a control 32P-labeled GAPDH cDNA (Clontech). Densitometric analysis was preformed using a AlphaImage 2000 imaging system (Alpha Innotech Corp.).

Southern Blot Analysis.
High molecular weight DNA was obtained from cultured cells and digested with HindIII for 5–7 h. Digestion products were separated by size on 0.8% nondenaturing agarose gels and transferred to GeneScreen Plus nylon membranes (NEN). The blots were prehybridized and then hybridized with 32P-labeled cDNA probes.

Construction of Sense and Antisense MT-II Expression Vectors.
A pair of primers, 5'-GATCCAACCGTCGTCTTCACT-3' and 5' -GCTCTATTTACAGGCAGCTGAGG-3', the sequences of which were taken from that of a published hamster cDNA (7) , were used to amplify a full-length MT-II cDNA by PCR. The amplified MT-II cDNA was cloned using a TA Cloning kit (Invitrogene) and then subcloned in sense orientation into the mammalian expression vector pcDNA3.1(+)/neo (Invitrogene). The entire sequence and its orientation were confirmed by DNA sequencing using a Sequenase Version 2.0 DNA Sequencing kit (Amersham). A second set of primers, 5' -AGTCTAGAACCGTCGTCTTCACTCG-3' and 5' -TGGTACCTGTCCGAAGCCTCTTTGC-3', containing XbaI and KpnI restriction sites, respectively, as underlined, were then used in an RT-PCR experiment to amplify a 187-bp fragment of MT-II cDNA. The amplified DNA was digested with XbaI and KpnI and directionally cloned in reverse orientation into similarly digested pcDNA3.1/zeo (+) vector DNA. Orientation was confirmed by restriction analysis and sequencing analysis.

DNA Transfection.
DNA transfection was carried out with the use of Lipofectin Reagent (Life Technologies, Inc.). Transfectants were selected in 400 µg/ml G418 (Sigma) for 7–10 days. Individual clones were isolated and analyzed for the expression of MT mRNA by Northern blot. Transfectants expressing antisense MT-II mRNA were identified by RT-PCR using the XbaI containing primer shown above and a T7 promoter-specific primer, 5'-TTATACGACTCACTATAGGG-3' located downstream of the transcription start site, resulting in a 240-bp product. The amount of MT expression in all clones was assessed by direct immunofluorescence staining using a MT-specific antibody, Cy3-Dako-MT, kindly provided by Dr. J. Lazo (University of Pittsburgh, Pittsburgh, PA). To permit comparisons of fluorescence intensity, all samples were processed simultaneously and photographed at 15 s of illumination.

Growth Assay.
Subconfluent cells (2 x 105) were harvested and seeded in a series of 25-cm2 flasks in FA medium. The medium was replaced every 3 days. Cells were harvested at 24-h intervals, and cell numbers were determined by counting under the microscope after staining with 0.4% trypan blue.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
MT-II Is Overexpressed in Cells Selected for Growth in 15 pM Folinic Acid.
One of the clones selected for survival in 15 pM FA medium, FA3, and its parent, DC-3F, were compared by mRNA differential display to identify genes whose expression level was affected by growth under conditions of severe folate restriction. One hundred twenty different combinations of primer sets made from three H-T11M anchored primers and 40 arbitrary primers, calculated to account for ~50% of mRNA transcripts in cells (8) , were used for RT-PCR. cDNA fragments representing 15 different mRNA transcripts were found to be reproducibly and differentially expressed between FA3 and DC-3F. These fragments were reamplified by PCR with the same sets of primers used to originally identify them and were used as probes for Northern blots of total cellular RNA. Two of these fragments (A1AP32a and C1R1) detected differential mRNA expression by this method. The remainder did not, presumably because they were either too short to be used for Northern blot probes or because their respective mRNAs are of low abundance and cannot be readily detected in total RNA preparations (8 , 9) . Both A1AP32a and C1R1 were sequenced, and a computer search of the GenBank and EMBL DNA databases revealed that C1R1 cDNA was homologous to the mouse BP75 gene (GenBank accession number AF084250), the function of which remains unknown. The A1AP32a sequence, however, shared >97% homology with the Chinese hamster MT-II gene.

To confirm that MT mRNA is overexpressed in FA3 cells, a full-length MT-II cDNA clone was obtained as described in "Materials and Methods" and used as a probe for a Northern blot analysis. The use of this probe does not allow reliable discrimination between MT-I and MT-II mRNAs that share ~80% sequence homology (7) . However, because the two genes are often coordinately expressed (10 , 11) , we chose not to attempt to differentiate between them. Hence, the results, shown in Fig. 1ACitation , reflect the contribution of both transcripts and confirm that MT mRNA is overexpressed in FA3 cells and in two other clones, FA7 and FA14, selected for growth in 15 pM folinic acid, as well.



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  		Fig. 1. Northern and Southern blot analysis of MT mRNA levels and gene copy number in cells selected for growth in 15 pM folinic acid. A, Northern analysis. Twenty µg of total RNA were denatured in formaldehyde/formamide, electrophoresed through a 1% agarose gel, and blotted onto nylon membranes. The membranes were hybridized with 32P-dCTP-labled MT-II cDNA probe and, after autoradiography, stripped and rehybridized with a [32P]dCTP-labeled ß-actin cDNA control probe. Lane 1, DC-3F; Lane 2, FA3; Lane 3, FA7; Lane 4, FA14. Overexposure of this blot shows a low level of MT mRNA expression in DC-3F (not shown). B, Southern analysis. Ten µg of HindIII-digested genomic DNA were separated on an 0.8% nondenaturing agarose gel, transferred to a nylon membrane, and hybridized with a [32P]dCTP-labeled MT-II cDNA probe. Lane 1, DC-3F; Lane 2, FA3; Lane 3, FA7; Lane 4, FA14. Based upon the restriction map of the hamster MT locus (23) , the 2.5-kb band represents the MT-II gene. Overexposure of this blot reveals an additional HindIII band of ~10 kb, which represents the MT-I gene, the copy number of which did not change.

 
FA3, FA7, and FA14 cells all amplify and overexpress the FR{alpha} gene.4 To determine whether folate depletion also results in MT gene amplification, genomic DNA was digested with HindIII and analyzed by Southern blots. As shown in Fig. 1BCitation , no evidence for amplification of MT genes was found in any of the three FA clones. Therefore, the up-regulation of expression did not result from an increase in gene copy number. Because either an increase in stability or an increase in the amount of transcription can elevate the steady-state level of any given RNA transcript, the half-life of MT mRNA was determined in both FA3 and DC-3F cells. After the addition of 5 µg/ml actinomycin D at time 0, the decrease in the amounts of MT mRNA and control GAPDH mRNA was measured at different times by Northern blots. The results (Fig. 2)Citation indicated that although there may be a small (<25%) increase in the half-life of MT mRNA in FA3 cells as compared with DC-3F, it cannot explain the 10-fold increase of MT mRNA noted in FA3. Hence, the increase in expression is largely due to a change in transcription.



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  		Fig. 2. Determination of MT mRNA half-life. Cells were treated with 5 µg/ml actinomycin D for 0, 3, 6, 8, or 24 h, and total RNA was isolated from each time point. Northern blot analysis was first carried out using a [32P-]dCTP-labeled MT-II cDNA probe. After autoradiography, the blots were stripped and reprobed with a similarly labeled GAPDH cDNA control probe (inset). Image analysis of the resulting autoradiograms was used to quantify each band and to measure the decay in signal intensity with time. The results are plotted as the percentage of the original MT mRNA remaining at the various time points. {blacksquare}, FA3 (FA media); FA3 cells were maintained in FA media with 15 pM folinic acid. {blacktriangleup}, FA3 (MEM/F12 media); FA3 cells were cultured in MEM/F-12 media for 7 days. {blacktriangledown}, DC-3F; DC-3F cells were maintained in MEM/F-12 media.

 
MT mRNA Expression in FA3, but not DC-3F, Is Sensitive to Media Folate Concentrations.
The overexpression of MT in all three FA clones indicated that folate depletion affects MT gene expression. To determine whether this effect was reversible, FA3 cells were plated in MEM/F12 and cultured for 7 days, at which time total RNA was prepared and analyzed for MT mRNA levels. These cells were then returned to FA medium, and after 7 days, their RNA was again analyzed. As determined by image analysis of the blot in Fig. 3ACitation , FA3 cells express at least 10-fold more MT mRNA than DC-3F. However, after 3 days in MEM/F12, the MT mRNA level started to decrease. This decrease continued, and at day 7, the expression level was comparable with that in DC-3F. Seven days after replating the cells in FA medium, MT mRNA expression returned to FA3 levels. In neither case did the half-life of MT mRNA change (Fig. 2)Citation . Hence, the steady-state level of MT mRNA in FA3 cells is directly sensitive to and inversely proportional with media folate levels and is transcriptionally controlled. DC-3F cells, however, show no such sensitivity, because culturing in FA medium did not affect the level of MT mRNA (Fig. 3B)Citation .



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  		Fig. 3. MT mRNA expression in FA3 cells, but not DC-3F, is sensitive to media folate concentration. A, MT mRNA expression level in FA3 cells grown in the media with different concentrations of folate. Lane 1, DC-3F; Lane 2, FA3 in FA media; Lanes 3–9, FA3 cells were put in MEM/F-12 media for 3, 6, 15, or 24 h or 3, 5, and 7 days, respectively; Lanes 10–12, FA3 cells grown in MEM/F-12 media for 7 days were put back to FA media for 3, 5, and 7 days, respectively. B, MT mRNA expression level in DC-3F cells grown in the media with different concentrations of folate. Lane 1, FA3; Lane 2, DC-3F cells in MEM/F-12 media; Lanes 3–5, DC-3F cells were put in FA media with 15 pM folinic acid for 3, 5, and 7 days, respectively; Lanes 6 and 7, DC-3F cells grown in FA media with 15 pM folinic acid for 7 days were put back to MEM/F-12 media for 3 and 7 days, respectively.

 
MT-II Overexpression Can Support DC-3F Cell Growth in 15 nM Folinic Acid.
The up-regulation of MT gene expression in FA3, FA7, and FA14 cells suggests either that overexpression of the protein product provides survival advantage or that it is just a phenomenon accompanying the low folate selection process. To address this issue, a full-length MT-II cDNA was cloned into the mammalian expression vector pcDNA3.1/neo(+) and transfected into DC-3F cells. A control vector without an insert was also prepared and transfected as well. Individual clones were isolated from populations of cells surviving after selection for 7–10 days in MEM/F12 containing 400 µg/ml G418 and then expanded in FA medium with 15 nM folinic acid. The resulting clones were analyzed by high stringency Northern blots for the expression of MT mRNA, and one of them, DCMT4, was found to expresses it to approximately FA3 levels (data not shown). Subsequent cell growth assays (Fig. 6)Citation showed that overexpression of the MT-II mRNA in transfectants, although unable to support growth in 15 pM folate, was able to do so when cells were cultured in 15 nM folate, conditions in which sham-transfected DC-3F cells are unable to sustain growth.



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  		Fig. 6. Effect of MT-II expression on DC3F cell growth in FA media. Growth curves were carried out as described in "Material and Methods" and in the presence of 5% dialyzed fetal bovine serum. The starting number of cells in each case was 2 x 105. A, cells were plated and maintained in FA media with 15 nM folinic acid. B, cells were plated and maintained in FA media with 15 pM folinic acid. DCMT4, MT-II cDNA transfectant; DCVE1, pcDNA3.1/neo(+) vector only transfectant. AS1 and AS5, antisense MT-II RNA expressing DCMT4 cell clones; bars, SD.

 
To demonstrate that the observed effect was directly attributable to MT-II overexpression, DCMT4 cells were transfected with an antisense MT-II construct in which Zeocin was used as the selectable marker. The double transfectant was found to express antisense MT-II RNA (Fig. 4)Citation and to express control levels of MT, as determined by immunohistochemistry using a MT-specific antibody (Fig. 5Citation , compare B and C with A). The double transfectant was unable to sustain growth in 15 nM folinic acid and displayed a growth curve similar to that of sham-transfected DC-3F (Fig. 6)Citation .



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  		Fig. 4. Reverse transcriptase/expression of antisense MT-II RNA in transfectants. PCR was used to demonstrate the expression of antisense MT-II RNA in transfectants. DCMT4 transfectants that overexpress MT-II RNA were retransfected with a second vector, pcDNA3.1/zeo(+) containing an MT-II cDNA insert cloned in antisense orientation to the cytomegalovirus promoter. Ten clones were analyzed, and two clones, AS1 and AS5, were found to express antisense RNA. Lane 1, 100-bp ladder; Lane 2, positive PCR control showing the expected 240-bp product obtained by using the antisense primer set described in "Materials and Methods" and the plasmid pcDNA3.1/neo (+) containing a full-length MT-II cDNA insert as substrate; Lane 3, RT-PCR product from DCMT4 transfectants. These cells do not express antisense MT-II RNA; Lanes 4 and 5, RT-PCR products from the double transfectant clones AS1 and AS5, showing the presence of antisense MT-II RNA.

 


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  		Fig. 5. MT-II expression levels in transfectants. Direct immunofluorescence microscopy was used to estimate MT-II expression levels. Samples were prepared simultaneously for each experiment, and all images were recorded after an exposure time of 15 s. Two independent experiments were carried out with cells plated on different days. Representative results are shown. A, DCMT4 transfectants; B, DC-3F cells sham transfected with pcDNA3.1/neo(+) without an insert; C, AS5 cells.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The depletion of intracellular folate pools that occurs by restricting media folate provides a useful experimental model with which to study changes in folate metabolism that must occur to maintain folate stasis and to determine the impact of such changes on related cellular and molecular events. Several studies have shown that up-regulation of FR{alpha} occurs when cells are cultured in low folate and that overexpression of the receptor enhances survival (1) . Similar results have been observed for FA3 cells selected for growth in folic acid-free media supplemented with 15 pM folinic acid (2) . However, FR{alpha} overexpression alone does not confer the complete FA3 phenotype, and transfectants do not display growth rates or cell cycle kinetics similar to those of FA3.5 To identify additional genes whose expression might be altered by growth in low folate and, therefore, contribute to the FA3 phenotype, mRNA differential display was used. MT-II was found to be one of the genes whose expression is up-regulated in cell lines selected for survival in 15 pM folinic acid. This unexpected association between MT-II overexpression and folate growth requirements provides a novel avenue for the study of the effects of this metal-binding protein on folate metabolism and perhaps on antifolate action as well. Although it has been reported that overexpression of MT does not provide resistance to the classical antifolate methotrexate (12) , resistance to nonclassical antifolates such as trimetrexate was not addressed. Moreover, it is not clear what effect the growth of cells in physiologically relevant folate concentrations i.e., nM levels of reduced folate, as opposed to the µM folic acid levels common to most standard tissue culture medium, would have on such studies. In addition, MT-II might act in conjunction with other overexpressed proteins to affect resistance. This has been suggested by the observation that DC-3F cotransfectants, which overexpress both FR{alpha} and MT-II, are three times more resistant to trimetrexate than transfectants that express similar levels of either protein alone.6

MT expression has been found to be inducible by a variety of factors including exposure to certain metals, phorbol esters, glucocorticoid hormones, and IFN as well as by inducers of the acute phase response (3 , 13) . Although folate does not act as an inducer of MT gene expression in FA3 cells, it is clear that the steady-state level of MT mRNA is inversely correlated with media folate levels, which directly affect the size of intracellular folate pools. Hence, in FA3, a decrease in the folate pool size is correlated with an increase in the steady-state level of MT mRNA. In addition to induction, one of the most frequent mechanisms found to be associated with MT gene overexpression has been gene amplification (13) . However, that was not found to be the case in any of the FA cell lines. Because the half-life of MT mRNA was found to be insensitive to media folate levels (Fig. 2)Citation , we conclude that the large increase in MT mRNA in these cells results from an increase in transcription.

The physiological function of MT remains elusive (5 , 14) . In addition to its role in zinc and copper absorption, MT has been reported to be involved in carcinogenesis and drug resistance (12 , 15) . Several recent studies indicate that expression of MT can prevent apoptosis and may constitute a protective mechanism that neutralizes apoptotic signals (16, 17, 18) . Our data are consistent with that notion and suggest that overexpression of MT-II in folate-depleted cells is not simply a phenomenon that accompanies selection. Rather, MT overexpression likely provides a growth advantage to FA3, FA7, and FA14 cells as it does for transfected DC-3F cells challenged for growth in 15 nM folinic acid (Fig. 4)Citation . In that case, it confers to DC-3F a growth rate similar to that of FA3, suggesting that MT overexpression alone can compensate for moderate folate depletion. The fact that it cannot do so in 15 pM folinic acid is because at such low external concentrations, DC-3F cells cannot obtain sufficient folate for survival (2) . This has been confirmed by the demonstration that transfectants, which overexpress FR{alpha} and show enhanced accumulation of folinic acid, survive in FA media, although they cannot maintain the FA3 growth rate.7 Whether DC-3F cells plated in 15 nM leucovorin die by apoptosis is under investigation.

The MT mRNA expression level in the three folate-depleted cell lines and its response to media folate concentrations (Fig. 3)Citation , coupled with the results of the transfection experiments (Fig. 6)Citation , suggest a relationship between MT expression and folate metabolism. However, the MT expression level in DC-3F cells was not found to be folate sensitive, and no major alteration of MT expression occurred in response to changes in media folate levels. It appears, therefore, that selection for survival in low folate has sensitized MT gene expression to folate status. Because hypomethylation has been reported to correlate with increased expression of MT (13) and is thought to be one of the consequences of growth in low folate (19) , it is attractive to consider the possibility that such an alteration has occurred in FA3 cells and is responsible for the up-regulation of MT mRNA expression. It has been suggested that overexpression of MT might provide a means of sequestering intracellular zinc from proteins that require it for activity, thus providing a novel form of regulation that has been proposed to be the physiological function of MT (20) . An intriguing example may be the folate conjugase enzymes (21) that, by removing glutamate residues, reduce the cellular retention times of intracellular folates and classical antifolates (22) . It has been reported that extracellular forms of the enzyme from human and pig brush border and chicken and rat pancreas require zinc for activity and that intracellular forms from the intestinal mucosa of several species do not (21) . However, conflicting results concerning the zinc requirements of the bovine liver enzyme exist (21) , and more information concerning the enzymes present in other tissue types is required before a complete understanding of the role played by these proteins in folate metabolism is clear. Nevertheless, sequestration of zinc from those forms of the enzyme that require it would be expected to protect polyglutamates and as a result, where the enzymes were intracellular, enhance folate retention, thus providing a growth advantage under conditions of folate stress. Clarification of these issues will provide new insights into the manner in which folate metabolism is regulated and impacts cell growth and antifolate sensitivity.


   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 by NIH Grant CA-49538 (to P. W. M.). Back

2 To whom requests for reprints should be addressed, at Biochemical Research Facility, University of Maryland, 108 North Greene Street, Baltimore, MD 21201. Back

3 The abbreviations used are: FR{alpha}, folate receptor {alpha}; MT, metallothionein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-PCR. Back

4 W. Zhu, M. A. Alliegro, and P. W. Melera. Overexpression of Chinese hamster folate receptor alpha is mediated by gene amplification and posttranscriptional regulation in CHL cells selected for growth under conditions of severe folate restriction, submitted for publication. Back

5 W-Y. Zhu and P. W. Melera, manuscript in preparation. Back

6 W-Y. Zhu and P. W. Melera, unpublished observations. Back

7 W. Zhu and P. W. Melera, manuscript in preparation. Back

Received 12/21/98. Accepted 7/19/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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