ICANCERRESEARCH54,5075-5080,October1, t994l

ABSTRACT

The genetic and epigeneticeffectsof nutritional folate deficiencywerestudied in two Chinese hamster ovary (CHO) cell lines. The CHO-AA8 cellline (hemizygousat the aprt locus)and CHO-UVS(DNArepair-deficientmutant of AA8) were cultured in Ham's F-12 medium or in customprepared Ham's F-12 medium lacking folic acid, thymidine, and hy

poxanthine. Cells cultured acutely in the folate deficient medium exhibitedinitial growth arrest, followed by massive cell death and DNA fragmentation into nucleosomal multimers characteristic of apoptosis. Althoughprolonged culture in the folate deficient medium was cytostatic and lethal

to the majority cells, minor subpopulations in both cell lines failed toinitiate cell death, exhibited phenotypic abnormalities, and adapted aselective growth advantage under marginal folate conditions. These “resistant― clones exhibited major alterations In deoxynucleotide pools maociated with an increase in mutant frequency at the aprt locus as detectedby resistance to cytotoxicity in 8-azaadenosine. The mutation frequency inthe DNA repair-deficient CHO.UV5 cells was @1OO-foldgreater than thatin the parental AA8clones,underscoring the Importance of DNArepairunder conditions of folate deficiency and nucleotide pool Imbalance. Theenhanced mutation frequency In the DNA repair-competent folate-defident CHO-AA8cells suggests that DNA repair activity Is less effectiveunder folate-deficientconditions. These results add to the accumulatingclinical and experimental evidence relating chronic folate deficiency togenomicinstability and carcinogenesis.

INTRODUCTION

Folate deprivation will induce aberrant DNA precursor metabolismbecause folate one-carbon groups are essential for the de novo synthesis of both purines and the pyrimidine thymidylate. On the basis ofsubstantial experimental evidence from both in vitro and in vivostudies, there can be little doubt that chronic imbalance in the ratio ofthe deoxynucleotide precursors for DNA synthesis is promutagenicand can modulate cellular sensitivity to the lethal and DNA-damagingeffects of known carcinogens (1—4).Several studies have demonstrated that the repair of spontaneous DNA damage is also significantly compromised under conditions of dN1T@ imbalance (5—7).Further, a “mutatorphenotype―can be both induced and reversed byappropriate manipulation of dNTP ratios (8, 9). Because the purineand pyrimidine dNTPs are the substrate for the DNA polymerases, thefidelity of DNA replication and repair synthesis is critically dependenton the correct balance and availability of the dNTP (10, 11). The sizeof the nuclear dNTP pools is extremely small and can sustain DNAsynthesis for only 30 s to 3 mm (1); therefore, alterations in dNTPprecursor metabolism may be an important mechanism to modulatecell cycle progression as well as the fidelity of DNA synthesis.

Recently, Borman and Branda (12) demonstrated that acute folate

Received 4/8/94; accepted 8/2/94.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 with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by American Cancer Society Research Grant CN-73B (J. J.) and a

National Center for Toxicological Research postgraduate fellowship (A. B.) administeredby Oak Ridge Institute for Science and Education through an interagency agreementbetween the Department of Energy and the United States Food and Drug Administration.

2 To whom requests for reprints should be addressed.

3 The abbreviations used are: dNTP, deoxynucleoside triphosphate; 8-AA, 8-azaade

nosine; CHO, Chinese hamster ovary; aprt, adenosine phosphoribosyltransferase; HPLC,high pressure liquid chromatography.

deprivation of CHO-Ki cells resulted in cell cycle arrest, cell death,and perturbation of cellular phenotype which could be modulated byavailability of folic acid, thymidine, and/or hypoxanthine. Despite thebiochemical stress associated with folate deprivation, a minor subpopulation of “resistant―cells adapted to survive under folate deficient conditions. The purpose of the present study was to confirm andextend these observations by determining whether chronic folatedeprivation in vitro would induce apoptotic cell death and whetherresistance to cell death (aberrant cell survival) was associated withdeoxyribonucleotide pool imbalance and/or genomic instability asevidenced by increased mutation frequency.

MATERIALS AND METHODS

Cells and Culture. The CHO cell lines AA8 and UV5 were originallyobtained from Dr. Larry Thompson (Lawrence Livermore National Laboratory,University ofCalifomia). The DNA repair-competent AA8 cellline is functionallyhemizygous at the aprt locus and is the parental cell line for the DNA repairdeficient cell line UV5. The cells were routinely maintained in standard Ham'sF12 medium (GIB@O-BRL, Grand Island, NY) supplemented with 50 @xg/migentamicin and 5% heat-inactivatedfetal bovine serum (Hyclone Laboratories,Logan UT) at 37°Cin a humidified 5% CO2 incubator. Folate-deficient Ham'sF12 mediumwas custompreparedby the vendorwithout folic acid, thymidine,orhypoxanthine in order to stress availability of precursors for both the de novo

pathway of nucleotide synthesis (folate derivatives) as well as precursors forsalvage pathway biosynthesis (thymidine, hypoxanthine). With the addition of 5%fetalbovineserum,thefolatecontentwas @—1 nMas determinedby microbiologled assay with Lactobacillus casei (13). Cells cultured in the custom prepareddeficient medium are hereafter referred to as folate-deficientcells. The controlcultureswere maintainedin completeHam's F12 mediumsupplementedwith 2.9p.M folic acid, 3 mM thymidine, and 30 mM hypoxanthine. Initial cell cultures were

seeded at a density of 5 X 10@cells/lO ml in T25 flasks in either complete orfolate-deficient medium. After a 2-day incubation, the cells were passaged into

fresh media, which was routinely changed 2 times a week until confluency wasreached. For subculture and experimental procedures, cell monolayers were detached by brief exposure to 0.25% trypsin at 37°C.Viable cells were counted in ahemocytometerby trypan blue dye exclusion. Detached (nonviable) cells weregently removed from the medium and enumerated separately. Cell death wasinitially confirmed by uptake oftrypan blue dye and subsequently by histology and

nucleosomalDNA fragmentationwith agamse gel electrophoresis.DNA Extraction and Analysisof DNA Fragmentation. DNA was cx

tracted from 3 x 106 cells by incubation in 300-@xldigestion buffer containing100 m@iNaC1, 10 mM Tris-HCI (pH 7.9), 25 mM EDTA, 0.5% sodium dodecylsulfate, and 0.3 mg/ml proteinase K for 15 h in a 50°Cshaker water bath. TheDNA was purified with phenol/chloroform/isoamyl alcohol (25:24:1) and theaqueous phase was extracted twice with diethyl ether to remove residual

phenol. The RNA was digested with 1 @xWmlDNase-free RNase for 1 h at37°C.The DNA was precipitated with 100% ice-cold ethanol, washed in 70%ethanol, dried in a Speed-Vac centrifuge, and dissolved in TE buffer (10 m@iTris-HC1, pH 7.9-1 mM EDTA). Approximately 1 gxg DNA was subjected toelectrophoresis on a 0.8% agarose minigel at 7 V/cm for 60 mm and visualizedwith ethidium bromide staining under UV illumination.

Trichloroacetic Acid Extraction and HPLC Determination of Deoxynucleotide Pools. Each of the cell lines were seeded into triplicate T25flasks in either control or folate-deficient Ham's F12 and grown until confluent. After 20 passages, monolayers were trypsinized and an aliquot of the cellsuspension was removed for cell count and viability determination. Aminimum of 5 x 106cells was required for deoxynucleotide HPLC detection.After centrifugation at 200 X g, 300 @xlof ice-cold 0.6 M trichloroacetic acid

5075

In Vitro Folate Deficiency Induces Deoxynucleotide Pool Imbalance, Apoptosis, and

Mutagenesis in Chinese Hamster Ovary Cells1

S. Jill James,2 Alexei G. Basnakian, and Barbara J. Miller

Division of Nutritional Toxicology. Food and Drug Administration. National Centerfor Toxicological Research, Jefferson, Arkansas 72079

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COMPLETEMEDIA FOLATE-DEFICIENTMEDIA

COMPLETE MEDIA FOLATE-DEFICIENT MEDIA

DEOXYNUCLEOTIDEPOOL IMBALANCE AND MUTAGENESIS

was added to the cell pellet followed by vigorous vortexing and 20 min on ice.The acidic supernatant was neutralized with 400-id Freon (trioctylaminetrichlorotrifluoroethane) and the aqueous upper phase was stored at —80°C,followed by direct injection into the HPLC. HPLC analyses were performed ona Beckman System Gold HPLC system consisting of a programmable solvent

module (Model 126) and wavelength detector (Model 168) with an Econosphere C18 reversed phase column (5 @xm,300 X 4.6 mm; Alltech Associates).Absorbance was monitored at 260 nm utilizing diode array detection andelution conditions as described previously (14). Quantitation of pool sizes was

accomplished utilizing individual calibration curves for each nucleotide andBeckman System Gold Software. The dNTP concentrations are expressed aspmol/@xgDNA.

Selection for aprr Mutants. Clones which had sustained mutations at theapri locus were selected by resistance to cytotoxicity in 8-AA using established methodology(15). Both the CHO-AA8 and AHO-UV5 surviving cloneswhich had been cultured for 20 passages were initiated at 5 X 10@cells/plate.Triplicate plates were cultured in parallel for determination of plating efficiency and mutant colony enumeration. Cells were exposed to medium con

taming 0.4 mM 8-AA and 5% dialyzed sera for 14 days to select for aprtmutants. Plating efficiency was determined after 8 days in culture. Care wastaken not to disturb the cells or change medium during the selection period toavoid generation of secondary colonies. Mutant clones surviving in 8-AA wereenumerated and the mutant frequencies were calculated by correcting the

observed mutant frequencies for the corresponding plating efficiencies.

RESULTS

Cell Proliferation, Cell Death, and Morphology. A comparisonof the initial cell growth rates in nutritionally complete Ham's F12media or in folate-deficient media is presented in Fig. 1. Cell proliferation was determined by quantitating the increase in viable cellnumbers (assessed by trypan blue exclusion) at 4-day intervals over a12-day incubation period. In the complete media, the rate of cellgrowth was virtually identical in both cell lines and was associatedwith viability >95% over the 12-day period. In contrast, cells culturedin the folate-deficient media were essentially cytostatic by day 4 afterthe first passage at day 2. Previous studies have established thatintracellular folate levels are reduced 10-fold in CHO cells after 5days in folate-deficient Ham's F12 medium (12). Growth inhibitionunder folate-deficient conditions was accompanied by a progressiveincrease in numbers of nonviable cells, followed by the emergence ofthe surviving clones. In Fig. 2, the relative rates of cell death incomplete media and in folate-deficient media indicate that the AA8

DAYS DAYS

U

Fig. 2. Cell death kinetics (increase in nonviable cell numbers) as assessed by trypanbluedyeuptakeof CHO-AA8andCHO-UV5celllinesculturedfor 12daysincompleteHam's F12 medium or in custom prepared folate-deficient Ham's F12 medium. All cellshad been passaged once after an initial 2 day exposure to complete or deficient medium.

cells were most sensitive to cell death under folate-deficient (thymineless) conditions. The fact that the DNA repair-deficient cell line,Uv5, was lesssensitiveto celldeathwasan unexpectedfindingsincethe DNA repair deficiency generally renders these cells hypersensitiveto DNA damage (16).

Morphological alterations in AA8 cells cultured in complete medium or in folate-deficient medium for 5 days are presented in Fig. 3,A and B. An increase in cell volume associated with cytostasis isapparent in the deficient cells. In Fig. 3, C and D, representativeclones adapted to the deficient conditions for 20 passages are shownto exhibit phenotypic polymorphisms including elongation and aspindle-shaped morphology or a rounded megaloblastoid morphology, often within the same culture.

Apoptosis and DNA Fragmentation. Detached (nonviable) cellswere gently aspirated from the medium on day 5 after the firstpassage. The attached cells were subsequently removed by gentle

trypsinization as described in “Materialsand Methods.―The DNAfrom approximately 106 viable (attached) and nonviable (detached)cells was extracted and subjected to electrophoresis at 7 V for 1.5 h ona 0.8% agarose gel (Sigma Chemical Co.). The DNA was visualizedby ethidium bromide staining under UV illumination and is presentedin Fig. 4. In Fig. 4, Lanes 1 and 2, the DNA from the nonviable AA8and UV5 cells, respectively, is shown to be fragmented into 180—200-base pair nucleosomal multimers characteristic of cell death byapoptosis. In Fig. 4, Lanes 3 and 4, DNA fragmentation in attached

viable cells is shown to be considerably less. However, it is apparentin Fig. 4, Lane 3, that DNA fragmentation is greater in the AA8 cellline, suggesting that this cell line is more sensitive to apoptosis thanthe UV5 cell line under folate-deficient conditions.

Deoxynucleotide Pools. The dNTP levels were not quantified during the initial phases of folate deprivation because of the possibleconfounding effects of dead and dying cells on dNTP pools. Therefore, dNTP measurements were only made on the stable proliferatingpopulations of clones adapted to survive in folate-deficient medium.In both AA8 and UV5 populations, adaptation to folate-deficientconditions was associated with significant increases in dCTP, dUTP,and dATP pools and decreases in the dTTP pool relative to cellsgrown in complete medium (Table 1). The dGTP pool was unchangedin Uv5 cells, and was undetectable in AA8 cells. The increase indUTP:dTTP ratio is consistent with a decrease in the folate-dependentmethylation of dTMP and a concomitant increase in the dUMP

5076

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Fig. 1. Growth kinetics (increase in viable cell numbers) of CHO-AA8 and CHO-UV5cell lines cultured for 12 days in complete Ham's F12 medium or in custom preparedfolate-deficient Ham's F12 medium. All cells were seeded to the respective complete ordeficient media for 2 days and passaged once prior to cell enumeration.

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DEOXYNUCLEOTIDEPOOLIMBALANCEAND MIJTAGENESI5

A B

D

Fig. 3. Morphology of CHO-AA8 cells during and after adaptation to folate-deficient medium (X 100). (A) CHO-AA8 cells maintained in complete Ham's F12 medium. (B) Growtharrest in folate-deficient medium after 8 days with one passage at day 2. Note megaloblastic appearance with homogenous morphology. (C and D) CHO-AA8 cells adapted tofolate-deficient media through 20 passages. Note cellular and colony polymorphism.

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precursor pool. The increase in dUTP would tend to inhibit thedeamination of dCFP to dUTP via a feedback mechanism (1) and mayhave contributed to the observed major increase in the dCTP pool anddCT'P:dTTP ratio. The decrease in d1TP might also have contributedto the increase in dC'FP via feedback up-regulation of dCTP synthesis(which is not dependent on folate availability) (17). An imbalance indNTP ratios has been shown to compromise the fidelity of the DNApolymerase (10) and induce mutagenesis in cultured cell lines (3, 18).

Mutagenesis. It was of interest to determine whether deoxynucleotide pool imbalance induced by marginal folate conditions wasassociated with enhanced mutation frequency in the surviving clones.

Mutation frequency was determined at the aprt locus because it is anonessential gene that has been shown to sustain a wide range ofmutations (15), because mutant colonies are easily selected by resistance 8-AA, and because the aprt hemizygous cell lines AA8 and UV5are hypersensitive to mutation detection at this locus (16). Cells thathave sustained inactivating mutations are unable to incorporate 8-AAvia the aprt salvage pathway and survive in its presence, whereasnonmutated cells with normal aprt activity will incorporate the toxic

analogue and die. In Table 2, the background mutation frequency inboth cell lines cultured for 20 passages in complete medium iscompared to the mutation frequency of the surviving clones after 20passages in low folate medium. The spontaneous mutation frequenciesin AA8 and UV5 cells grown in complete medium were not significantly different and approximated the established mutation frequencies for those CHO cell lines (16). In the AA8 and UV5 clones derivedfrom cells adapted to low folate conditions, the mutation frequency

was significantly increased. The UV5 cell line had the highest numberof mutant colonies, which was approximately 100-fold greater thanthat in the AA8 cells. The @-100-fold increase in mutation frequencyin the UV5 cells relative to the AA8 cells most likely reflects the fact

that the UV5 cell line is defective in the incision step of DNA excisionrepair and has accumulated significant unrepaired DNA lesions underconditions of marginal folate availability. The difference in mutationfrequency between the DNA repair-competent and DNA repair-deficient cell lines underscores the importance of normal DNA repaircapacity under conditions of folate deficiency and dNTP pool imbalance. The fact that folate deficiency increased mutation frequency inthe DNA repair-competent AA8 cells would suggest that normal DNArepair capacity was inadequate under these conditions.

DISCUSSION

Experimental, clinical, and epidemiological evidence has been accumulating in the last decade to indicate that inadequate dietary folatemay predispose to increased risk of certain types of human cancer (19,20). A role for localized folate deficiency in the etiology of humanepithelial cell cancer was indirectly implied by the significant reversalof premalignant dysplasia in cervical (21), bronchial (22), and epithehal cells and the decreased incidence of preneoplasic lesions incolonic cells (23) following oral folic acid supplementation. In epidemiological studies, low folate status has been associated with increased risk of colorectal adenoma (24), rectal cancer (25), cervical

neoplasia (26), HPV infection (27), and esophageal cancer (28).

5077

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DEOXYNUCLEOTIDEPOOL IMBALANCE AND MUTAGENESIS

In the present study, the spectrum of dNTP alterations in theadapted AA8 and UV5 cells are qualitatively similar to thosepreviously observed for dCMP deaminase-deficient cells (38). Increases in dCTP, dUTP, and dATP with concomitant decreases in

dTFP and dGTP have also been observed with cell growth underthymineless conditions (34). The increase in dCTP pool may reflect

the fact that it is the only dNTP that is not dependent on folateone-carbon transfer and that dCTP synthesis is up-regulated by adecrease in dTTP levels. An imbalance in the dCl'P:dTFP ratio hasbeen shown to have marked effects on rates of spontaneous mutagenesis predominantly due to base pair substitutions caused by misincorporation of the nucleotide precursor in excess (next nucleotide effect)(39). Recent studies have documented the misincorporation of uracilinto DNA of folate and B12-deficient HL6O cells (40) and also intoDNA of human bone marrow cells (41). The substitution of uradil forthymidine would not be expected to influence adenine base pairing;however, the efficient action of uracil glycolsylase would create apotentially mutagenic abasic site. Thus, the base composition of theDNA synthesized under these conditions may also be indirectly altered by a decrease in the folate-dependent methylation of dUMP todTMP.

The fact that the DNA repair-deficient cell line UV5 was found to beless sensitiveto cell death under folate-deficient(thymineless)conditionswas an unexpectedfindingsince DNA repairdeficiencygenerallyrendersthese cells hypersensitive to DNA damage (16). A possible explanationfor the decreased rate of cell death (Fig. 2) and the decrease in earlyapoptotic DNA fragmentation (Fig. 4, Lane 4) in UV5 cells is that thedefect in the incision endonuclease that characterizes the DNA repairdeficiency may be functionally related to the defect in the Ca/Mgdependentendonucleasethat fragments the DNA during apoptosis.Consistent with this possibility, the total Ca@ @-/Mg@k-dependent endonu

clease activity was found to be significantly lower in repair-deficientUV5 cells compared to AA8 cells (42).

It should be noted that the concentration of folate in the standardHam's F12 medium is 2.9 ELM.Relative to circulating human plasmalevels of 10—50flM,it would appear that folate exposure in the controlcultures was supraphysiological. However, it should be emphasizedthat the mechanisms by which the cell maintains intracellular folatehomeostasis are complex and not completely understood. For examplc, in humans, tissue (cell) concentrations of folate have been shownto be 3 orders of magnitude higher than circulating plasma levels (43).Further, the homeostatic maintenance of intracellular folate concentrations appeared to be remarkably constant despite a 40-fold exper

imental increase in normal plasma folate levels (44). Thus, an increasein exogenous folate concentration may not raise intracellular levels in

a dose-response manner at higher concentrations. Kamen et aL (43)have further shown that cells exhibit saturable receptor binding of5-methyl tetrahydrofolate, with maximum binding at 20 nr@iextracellular folate (within physiological limits). Taken together, the existingevidence would suggest that the folate concentration in Ham's F12medium is clearly supraphysiological but that cells may be able toregulate intracellular concentration of folate within a narrow marginregardless of the exogenous concentration.

Cellular adaptation to severe biochemical stress after an initialphase of high mortality has been observed in numerous experimentalmodels including anti-folate drug exposure (45, 51). A mechanism forcell survival under conditions of severe folate deprivation has beenrecently elucidated by Matsue et a!. (46). Cells that express a uniquehigh affinity folate membrane receptor are capable of transporting5-methyl tetrahydrofolate by the process of potocytosis and theysurvive to proliferate at folate levels

Table 1Deoxynucleotide poolsin CHO celLs after 20 passages in conzplete Ham ‘sF12 medium(C) orfolate-deficient Ham ‘sF12 medium(FDfpmolIg@g

DNA―IATPdCFP/dTFPdUIP/d1TPdCTPdUTP

dlTPdGTPCHO-AA8C

medium93 ±0.32.9 ±0.7 5.6 ±0.11.7 ±0.20.6 ±0.11.70.5FDmedium22.0 ±l.l'@4.5 ±03d @e1.7 ±O.8'@>22>4.5

6.5±0.71.6±0.10.4±0.10.90.51.5±0.2@1.5 ±0.10.8 ±0.2c143.4

Table 2 Generation time and mutation frequency in CHO cell lines after 20 passagesin complete Ham's F12 medium (C) or in folate-deficien: Ham's F12 medium (FD)

aones of CHO-AA8andUV5cellswhichhadadaptedtosurvivein lowfolateRPM!1640 were seeded in triplicate plates for determination of plating efficiency and mutantcolony formation. Cells were exposed to medium containing 0.4 mat 8-azaadenine and 5%dialysed serum for 14 days. Mutant clones surviving in AA8 were enumerated and themutation frequencies were calculated by correcting the observed mutant colonies for thecorresponding plating efficiencies.

Cell lineMediumGenerationtime

(h)Mutationfrequency

(X10CHO-AA8C

FD20.5 37.71.7±1.4

25.0 ±8.0CHO-UV5C

FD18.0 32.90.6 2800 ±270

DEOXYNUCLEOTIDEPOOL IMBALANcE AND M1.TFAGENESIS

CHO-UV5Cmedium 6.1±1.0 3.0±0.7FDmedium 21.0±1.6'@ 51@01d

a@ medium is deficient in folic acid, thymidine, and hypoxanthine.

b Mean ± SEM from 2—3 separate experiments.

C p < o.oi relative to control.

d p < 0.05 relative to control.

e@ not detectable.

REFERENCES

1. Meuth, M. The molecular basis of mutations induced by deoxynucleotide triphosphatepool imbalances in mammalian cells. Pip. Cell Res., 181: 305-316, 1989.

2. Kunz, B. A. Mutagenesis and deoxynucleotide pool imbalance. Mutat. Res., 200:133—147,1988.

3. Mattano, S. S., Palella, T. D., and Mitchell, B. S. Mutations induced at the hypoxanthine-guanine phosphoribosyltransferase locus of human T-lymphoblasts by perturbations of purine deoxyribonucleoside triphosphate pools. Cancer Res., 50:4566—4571,1990.

4. Kunz, B. A. Genetic effects of deoxyribonucleotide pool imbalances. Environ. Mutagen., 4: 695—725,1982.

5. Holliday, R. Aspectsof DNA repair and nucleotidepool imbalance.Basic Life Sci.,31: 453—460,1985.

6. Gobs, B., and Malec, J. Enhancement of methotrexate-induced growth inhibition, cellkilling and DNA lesions in cultured L5178 cells by the reduction of DNA repairefficiency. Biochem. Pharmacol., 38: 1743—1748,1989.

7. Snyder, R. D. Consequences ofthe depletion ofcellular deoxynucleoside triphosphatepoolson theexcision-repairprocessinculturedhumanfibroblasts.Mutat.Rca.,200:193—199,1988.

8. Newman, C. N., and Miller, J. H. Mutagen-induced changes in cellular deoxycytodinetriphosphate and thymidine triphosphate in Chinese ovary cells. Biochem. Biophys.Res. Commun., 114: 34—40,1983.

9. Weinberg, G., Ullman, B., and Martin, D. W. Mutator phenotypes in mammalian cellmutants with distinct biochemical defects and abnormal deoxyribonucleoside triphosphate pools. Proc. Nat!. Acad. Sd. USA, 78: 2447—2451,1981.

10. Das, S. K., Kunkel, T. A., and Loeb, L A. Effects of altered nucleotide concentrationson the fidelity of DNA replication. Basic Life Sci., 31: 117—126,1985.

11. Haynes, R. H. Molecular mechanisms in genetic stability and change: the role ofdeoxyribonucleotidepoolbalance.In: F. S.deSerres(ed), GeneticConsequencesofNucleotide Pool Imbalance, pp. 1—23,New York: Plenum Publishing Corp., 1985.

12. Borman, L S., and Brands, R. F. Nutritional folate deficiency in Chinese hamsterovary cells. I. c@haracterizationof the pleiotropic response and its modulation bynucleic acid precursors. J. Cell. Physiol., 140: 335—343,1989.

13. Newman, E. hi., and Tsai, 3. F. Microbiological analysis of percent-formyltetrahydrofolic acid and other folates using an automatic 96-well plate reader. Anal.Biochem., 154: 509—515,1986.

14. Cross, D. R., Miller, B. J., and James, S. I. Asiinplified HPLC method for quantifyingribonucleotides and deoxynucleotides in cell extracts and frozen tissues. Cell Proliferation, 26: 327—336,1993.

15. Goncalves, 0., Drobetsky, E., and Meuth, M. Structural alterations of the apri locusinduced by deoxyribonucleotide triphosphate pool imbalances in Chinese hamsterovary cells. Mol. Cell. Biol., 4: 1792—1799,1984.

16. Thompson, L H., Brookman, K. W., Dillehay, L E., Mooney, C. L, and Carrano,A. V. Hypersensitivity to mutation and sister chromatid exchange induction in CHOmutants defective in incising DNA containing UV lesions. Somatic Cell Genet., 8:759—773,1982.

17. Haynes, R. H., and Kunz, B. A. The influence of thymine nucleotide depletion ongenetic stability and change in eukaryotic cells. Current Sci., 55: 1—11,1986.

18. Kunz, B. A., and Kohalmi, S. E. Modulation of mutagenesis by deoxyribonucleotidelevels. Annu. Rev. Genet., 25: 339—359,1991.

19. Rosenberg, I. H., and Mason, J. B. Folate, dysplasia, and cancer. Gastroenterology,97:502—503,1989.

20. Eto, I., and Krumdieck, C. L Role of vitamin B12 and folate in carcinogenesis. In:L A. Poirier, P. M. Newberne, and M. W. Pariza (eds.), Essential Nutrients inCarcinogenesis, pp. 313-330. New York: Academic Press, 1986.

21. Butterworth, C. E., Hatch, K. D., Gore, H., MealIer, H., and Krumdieck, C. LImprovement in cervical dysplasia with folic acid therapy in users of oral contraceptives. Am. J. Gin. Nutr., 35: 73—82,1982.

22. Heimburger, D. C., Alexander, B., Birch, R., Butterworth, C. E., Bailey, W. C., andKrumdieck, C. L Improvement in bronchial squamous metaplasia in smokers treatedwith folate and vitamin B12. JAMA, 259: 1525—1530,1988.

23. Lashner, B. A., Heidenreich, P. A., Su, G. L, Kane, S. V., and Hanauer, S. B. Effectof folate supplementation on the incidence of dysplasia and cancer in chroniculcerative colitis. A case-control study. Gastroenterology, 97: 255—259,1989.

24. Giovannucci, E., Stampfer, M. J., Colditz, G. A., Rimm, E. B., Trichopoulos, D.,

phosphoribosylpyrophosphate pools for de novo nucleotide ring synthesis (48). Severe folate deficiency also leads to elevated levels ofS-adenosylhomocysteine (49), which can be catabolized to adenosineand inosine to serve as potential sources of purse salvage precursors.Such adaptive responses may offer a partial explanation for the abilityof the minor subpopulation of cells in the present study to establishstable growth kinetics under folate-deficient conditions.

The clonal adaption theory of carcinogenesis postulates that the rarepremalignant cell acquires the ability to adapt and survive underconditions of severe metabolic stress, which are mitoinhibitory orlethal to the majority of cells (50). A similar sequence of events wasobserved in the present study under the in vitro metabolic stress offolate deprivation; a minor subpopulation of cells adapted to surviveconditions which were cytostatic and lethal to the majority of cells.

The biochemical stress induced by folate deficiency would appear tobe deoxynucleotide DNA precursor imbalance secondary to the requirement for folate in de novo purine and thymidylate biosynthesis.

In this study we provide evidence that nutritional folate deficiencycan induce mutagenesis in a highly sensitive in vitro model and offera possible mechanism for genetic lesions associated with folate deprivation. In previous in vivo studies, we have shown that dietary folatedeficiency produces DNA strand breaks (51), abnormal DNA repairactivity (52), and dNTP pool imbalance in rat lymphocytes (53).

Further, phytohemagglutinin-stimulated lymphocytes cultured underfolate-deficient conditions also exhibit significant alterations in dNTPpools, which are associated with abnormal DNA synthesis and cellcycle progression (54). Recently, Branda has provided additionalevidence that in vitro folate deficiency potentiates DNA strand breaksinduced with alkylating agents by limiting DNA repair efficiency inCHO cells (55). Whethersimilar biochemical and molecularmechanisms contribute procarcinogenic effects with folate deficiency in vivowill require continued investigation with dietary models.

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DEOXYNUCLEOTIDEPOOL IMBALANCE AND MUTAGENESIS

Rosner, B. A., Speizer, F. E., and Willett, W. C. Folate, methionine and alcohol intakeand risk of colorectal adenoma. i. NatI. Cancer Inst., 85: 875—883,1993.

25. Freudenheim, J. L, Graham, S., Marshall, J. R., Haughey, B. P., Cholewinski, S., andWilkinson, G. Folate intake and carcinogenesis of the colon and rectum. Int. i.Epidemiol., 20: 368—374, 1991.

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1994;54:5075-5080. Cancer Res S. Jill James, Alexei G. Basnakian and Barbara J. Miller Ovary CellsImbalance, Apoptosis, and Mutagenesis in Chinese Hamster

Folate Deficiency Induces Deoxynucleotide PoolIn Vitro

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