Proc. Natl. Acad. Sci. USAVol. 93, pp. 4045-4050, April 1996Genetics

Increased cytosine DNA-methyltransferase activity istarget-cell-specific and an early event in lung cancer

(alveolar type II cell/A/J mouse/susceptibility/biomarker)

STEVEN A. BELINSKY*t, KRISTEN J. NIKULA*, STEPHEN B. BAYLINt, AND JEAN-PIERRE J. ISSAt*Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, NM 87185; and tJohns Hopkins University, 424 North Bond Street,Baltimore, MD 21231

Communicated by Allan H. Conney, Rutgers University, Piscataway, NJ, January 2, 1996 (received for review October 23, 1995)

ABSTRACT The association between increased DNA-methyltransferase (DNA-MTase) activity and tumor develop-ment suggests a fundamental role for this enzyme in theinitiation and progression of cancer. A true functional role forDNA-MTase in the neoplastic process would be further sub-stantiated if the target cells affected by the initiating carcin-ogen exhibit changes in enzyme activity. This hypothesis wasaddressed by examining DNA-MTase activity in alveolar typeII (target) and Clara (nontarget) cells from A/J and C3Hmice that exhibit high and low susceptibility, respectively, forlung tumor formation. Increased DNA-MTase activity wasfound only in the target alveolar type II cells of the susceptibleA/J mouse and caused a marked increase in overall DNAmethylation in these cells. Both DNA-MTase and DNA meth-ylation changes were detected 7 days after carcinogen expo-sure and, thus, were early events in neoplastic evolution.Increased gene expression was also detected by RNA in situhybridization in hypertrophic alveolar type II cells of carcin-ogen-treated A/J mice, indicating that elevated levels ofexpression may be a biomarker for premalignancy. Enzymeactivity increased incrementally during lung cancer progres-sion and coincided with increased expression of the DNA-MTase gene in hyperplasias, adenomas, and carcinomas.Thus, these results indicate that early increases in DNA-MTase activity are strongly associated with neoplastic devel-opment and constitute a key step in carcinogenesis. Thedetection of premalignant lung disease through increasedDNA-MTase expression and the possibility of blocking thedeleterious effects of this change with specific inhibitors willoffer new intervention strategies for lung cancer.

Evidence is emerging to suggest a fundamental role forincreased activity by DNA-methyltransferase (DNA-MTase),the enzyme that catalyzes DNA methylation at CpG sites (1),in the initiation and progression of cancer. First, DNA-MTaseactivity increases incrementally over the progressive stages ofhuman colon cancer (2). Second, overexpression of the murineDNA-MTase gene in NIH 3T3 cells results in transformation(3). Third, lowering DNA-MTase activity in mice geneticallypredisposed to formation of colonic adenomas markedly de-creases the frequency of these lesions (4). The mechanismsunderlying the association of increased DNA-MTase activitywith tumorigenicity are not fully understood. However, re-gional hypermethylation events involving normally unmethy-lated CpG islands in promoter regions have now been associ-ated with the inactivation of several tumor suppressor genes(5-10). In addition, methylated cytosines are hotspots for C toT mutations (11), and under certain conditions, the activity ofDNA-MTase itself can result in this mutational transition (12).

If the associations between increased DNA-MTase activityand tumor development indicate a true functional role for the

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enzyme in this process, then target cells affected by theinitiating carcinogens should show a significant increase inenzyme activity. The present investigation addresses this ques-tion by using a mouse model of lung carcinogenesis in whichspecific candidate target cell populations can be studied.Furthermore, inbred strains of mice with differing suscepti-bilities to carcinogen-induced neoplasia are examined in orderto define relationships between target cell response, changes inDNA-MTase activity, and tumorigenesis. The murine modelsused for this investigation are the A/J mouse, which is sensitiveto the development of lung tumors (13) that arise from alveolartype II cells (14), and the C3H mouse, which has a lowersusceptibility for tumor formation (15). Thus, the purpose ofthis investigation was to determine the effect of in vivocarcinogen exposure on DNA-MTase activity in target (alve-olar type II) and nontarget (Clara) cells isolated from A/J andC3H mice and to quantitate DNA-MTase activity duringtumor progression in the A/J mouse lung. Our results providefurther evidence that early increases in DNA-MTase activityare directly associated with neoplastic development and mayconstitute a key step in carcinogenesis.

MATERIALS AND METHODSAnimal Treatment, Cell Isolation, and S Phase Determina-

tion. A/J and C3H mice (6-8 weeks old, The Jackson Labo-ratory) were treated three times (every other day, 50 mg/kg,i.p.) with 4-methylnitrosamino-l-(3-pyridyl)-l-butanone(NNK, Chemsyn Science Laboratories, Lenexa, KS) dissolvedin saline or with saline alone (0.2 ml). A/J mice were sacrificed1, 3, 7, and 14 days after treatment, while C3H mice andsaline-treated mice were sacrificed 7 days after treatment.Small cells (endothelial cells and lymphocytes), alveolar typeII, and Clara cells were obtained in separate aliquots bycentrifugal elutriation following protease digestion of the lungs(16), which were pooled from 18 mice for isolation of cells.Three to five groups of mice comprised each time point. TypeII and Clara cell preparations used for analysis ofDNA-MTaseactivity and DNA methylation levels were 73 ± 2% and 76 ±4% pure, respectively. Small cells and macrophages were themajor contaminating cells within the alveolar type II and Claracell preparations, respectively. A sample of the isolated lungcells (1 x 106 cells) was fixed in 4% paraformaldehyde andincubated with RNase, and the DNA was stained with pro-pidium iodide. The percent of cells in S phase was determinedby a flow cytometric analysis.Lung tumors were induced in A/J and C3H mice (50 mice

per strain) by treatment of mice three times a week for 7 weekswith NNK (50 mg/kg, i.p.). White to cream-tan nodules werereadily evident on and just below the pleural surface of thecollapsed lung after mice were euthanized. All nodules werecounted before inflation of the lung with 4% buffered para-

Abbreviations: DNA-MTase, DNA-methyltransferase; NNK, 4-meth-ylnitrosamino-l-(3-pyridyl)-l-butanone.

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Proc. Natl. Acad. Sci. USA 93 (1996)

formaldehyde or prior to microdissection. Hyperplasias, ade-nomas, and carcinomas constituted the surface lesion counts.The validity of enumerating surface nodules in mouse lungs toestimate tumor multiplicity has been described previously (17).A/J mice were sacrificed 20, 28, 36, 44, and 52 weeks (10 miceper time point) after initiation of treatment, pulmonary nod-ules were microdissected from the right apical or cardiac lobefrom 10 mice at all time points excluding the 20 week point, andthe remaining lung lobes were inflated with 4% paraformal-dehyde fixative. C3H mice were sacrificed 20, 28, 52, 60, and68 weeks after initiation of treatment, and nodules weremicrodissected at the 68 week time point.DNA-MTase Enzyme Assay. The assay developed by Adams

et al. (18) was modified to determine DNA-MTAse activity.Cell lysates containing 5 ,ug of protein were incubated for 2 hat 37°C with a deoxyinosine-deoxycytosine template and 3H-labeled S-adenosylmethionine (Amersham). Reactions were

stopped, protein was extracted, and the deoxyinosine-deoxycytidine template was recovered by ethanol precipita-tion. RNA was removed by resuspension of the precipitates inNaOH; DNA was spotted on Whatman filters, dried, and thenwashed with trichloroacetic acid (5%) followed by 70% etha-nol. Filters were placed in scintillation mixture, and DNA-MTase activity was determined by scintillation counting. Re-sults were expressed as dpm/,ug of protein; all assays were

performed in duplicate. Limits of detection were 20 dpm abovebackground levels (background levels were determined in assaysin which the deoxyinosine-deoxycytosine template was omitted).Statistical analyses were performed using Student's t test.DNA Methylation Level. A methyl-accepting assay (3) was

used to determine the methylation status ofDNA isolated (19)from alveolar type II cells from A/J and C3H mice 7 days aftercessation of carcinogen treatment. DNA (150 ng) was incu-bated at 37°C for 4 h with 4 units of M.SssI CpG methylase(New England Biolabs) in the presence of 3H-labeled S-adenosylmethionine and nonradioactive S-adenosylmethi-onine. Reactions were stopped by adding nonradioactive S-adenosylmethionine, spotted on Whatman filters, dried,washed with trichloroacetic acid (5%) followed by 70% etha-nol, and placed in scintillation mixture for assay of radioac-tivity. Reactions without either DNA or enzyme added wereincluded as background controls; counts in these samplesnever exceeded 2-5% of those for test samples. All sampleswere analyzed in duplicate, and values were obtained as

dpm/ng ofDNA. DNA methylation levels are inversely relatedto the counts obtained; that is, a decrease in counts indicatesan increase in methylation. Therefore, to express the data interms of percentage of increase in methylation, counts ob-tained in type II cells from NNK-treated mice were divided bycounts from saline controls. The inverse of the product wasthen determined and expressed as a percentage of salinecontrol. Statistical analyses were performed using Student's t-test.DNA-MTase Gene Expression. A nonradioactive, in situ

hybridization technique was developed to examine DNA-MTase gene expression during lung tumor progression. Anantisense oligonucleotide probe complementary to a 24-nucleotide sequence spanning bases 1811-1834 of the murineDNA-MTase gene (14) and the corresponding sense probewere commercially synthesized (Research Genetics, Hunts-ville, AL). A biotinylated extender region [seven repeatedunits of d(TAG) with six biotin moieties] was coupled to the3' end (20) during synthesis. Serial lung sections (5 ,tm) fromA/J mice sacrificed 52 weeks after initiation ofNNK treatmentwere cut and mounted on charged glass microscope slides(ProbeOn Plus, Fisher Scientific). The slides were deparaf-finized, acetylated, and treated with pepsin as described byRadinsky et al. (21), except that the pepsin treatment was 30min at 37°C. Endogenous alkaline phosphatase was blockedwith Endoblocker (Biomedia, Foster City, CA). As a control,serial sections were incubated for 1 h at 37°C with RNase A

type 1A (Sigma) at 1 mg/ml and RNase T1 (Sigma) at 25units/,ld in 50 mM Tris HCl (pH 7.5). Slides not pretreatedwere incubated in Tris for the same time. All slides wereincubated for 1 h at 37°C with DNase I (Gibco/BRL) at 1unit/,tl in 50 mM Tris-HCl, (pH 7.5)/7 mM MgCl2. Theantisense and sense probes (1 uLg/,l), diluted 1:200 in ProbeDiluent (Research Genetics), and Probe Diluent alone (con-trol for endogenous alkaline phosphatase) were applied toserial sections that were incubated at 100°C for 10 min, cooledfor 3 min at room temperature, and incubated for 18 h at 40°C.The slides were washed with Probe Wash (Biomedia) contain-ing 25% acetone three times at 37°C, incubated with alkalinephosphatase avidin conjugate (Biomedia) for 30 min at 40°C,rinsed with 50 mM Tris buffer (pH 7.6), rinsed with alkalinephosphatase enhancer (Biomedia) for 30 s, and incubated withthe Fast Red chromogen (Biomedia) according to the manu-facturer's instructions for 20 min at 40°C. The slides wererinsed with Tris buffer, counterstained with aqueous hema-toxylin, covered with Crystal Mount (Biomedia), and exam-ined by light microscopy.

RESULTSCell- and Strain-Specific Increases in DNA-MTase Activity

Following Carcinogen Exposure. Endogenous activity ofDNA-MTase was similar between alveolar type II and Claracells from A/J mice, while enzyme activity was greater in thesmall cell fraction (Table 1). One day following treatment withthe tobacco-specific nitrosamine, NNK, DNA-MTase activitywas increased 2-fold (P < 0.05) in alveolar type II cells fromA/J mice (Table 1). DNA-MTase activity continued to in-crease in alveolar type II cells for at least 1 week. No effect onDNA-MTase activity following NNK treatment was observedin Clara cells or small cells. Moreover, enzyme activity was fourtimes greater in alveolar type II cells than Clara cells 1 weekafter cessation ofNNK treatment (Table 1). The percentage ofalveolar type II, Clara, and small cells in S phase 3, 7, or 14 dayspost NNK treatment did not differ from cells isolated fromunexposed mice (data not shown), negating a role for cellreplication in the increase in DNA-MTase activity.The marked increase in DNA-MTase activity observed in

alveolar type II cells from A/J mice suggests that the changein activity of this enzyme following carcinogen exposure mightpredict susceptibility for lung cancer. This hypothesis wasexamined by determining DNA-MTase activity in lung cellsisolated from the C3H mouse, a strain that has a lowersusceptibility for lung tumor development as compared withthe A/J mouse (15). Endogenous levels of DNA-MTaseactivity did not differ between alveolar type II and Clara cellsfrom A/J and C3H mice (Table 2). Cells were isolated fromC3H mice 7 days after carcinogen treatment, the time at whichalveolar type II cells in the A/J mice exhibit maximal changesin DNA-MTase activity. However, in contrast to A/J mice,DNA-MTase activity was not increased in alveolar type II cellsfrom C3H mice following carcinogen exposure (Table 2).Changes in DNA Methylation in Alveolar Type II Cells. The

effect of increased DNA-MTase activity in alveolar type II

Table 1. Effect of NNK treatment on DNA methyltransferaseactivity in lung cells from A/J mice

Methyltransferase activity, dpm/,ug of proteinCell type Endogenous la 3a 7a 14a

Type II 36 ±7 77 ± 20* 84 ± 7t 109 ± 14t 66 ± 13*Clara 37 ± 2 23 ± 6 42 + 14 26 + 14 36 ± 13Smalll cell 84 13 86 21 88 ± 4 76 21 73 + 20

Values are the mean ± SEM from three to five cell isolations pertime point. *, P < 0.05; t, P < 0.01 with respect to control.aDays after NNK treatment.

4046 Genetics: Belinsky et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 4047

Table 2. DNA methyltransferase activity in type II and Clara cellsfrom A/J and C3H mice 7 days after cessation of NNK treatment

Methyltransferase activity, dpm/,tg of proteinType II cells Clara cells

Mouse Endoge- Endoge-strain nous NNK nous NNK

A/J 36 + 7 109 + 14* 37 + 2 26 + 14C3H 39 ± 3 45 + 6 21 - 7 23 ± 7

Values are mean ± SEM from three to five isolations per time point.Data for A/J mice are from Table 1. *, P < 0.01 as compared withendogenous methyltransferase activity.cells was determined by a DNA methyl-accepting assay inwhich the capacity of cellular DNA to undergo in vitromethylation is inversely proportional to its endogenous meth-ylation level. An 85% increase (P < 0.01) in DNA methylationlevels, indicating marked hypermethylation (Table 3), was seenin alveolar type II cells isolated from A/J mice 7 days aftertreatment with NNK. In contrast, genomic DNA methylationlevels were not altered significantly in alveolar type II cellsfrom C3H mice after carcinogen treatment.Tumor Development and Multiplicity in A/J and C3H Mice.

Previous carcinogenicity studies with C3H mice (22) have onlyexamined tumor multiplicity 72 weeks after treatment withNNK. Therefore, in order to more accurately define differ-ences in susceptibility between A/J and C3H mice, lung tumormultiplicity was determined by serial sacrifice of mice over 68weeks following initiation of carcinogen exposure. Twentyweeks after NNK treatment was initiated, approximately 28nodules were present on the surface of lungs from A/J mice,while no nodules were evident on the surface of C3H lungs(Table 4). The number of surface nodules increased to ap-proximately 48 per A/J lung over the next 16 weeks. Pulmo-nary nodules in the C3H mouse arose between the 28 and 52week sacrifice points; approximately 10 nodules per lung weredetected at 52 weeks.

Increased DNA-MTase Activity During Tumor Progression.The mass distribution of pulmonary nodules analyzed fromA/J mice varied from 0.3 to 5.0 mg. Based on the establishedtumor progression model (14), nodules with the smaller mass(0.3-0.5 mg) were predominantly hyperplasias and small ad-enomas; adenomas and carcinomas were most prevalent in the0.6-1.5 and 3.1-5.0-mg size ranges, respectively. DNA-MTaseactivity for the various nodules grouped by size is shown in Fig.1. The enzyme activity increased in direct proportion to thesize of the nodules from 144 dpm/,Lg to >300 dpm/,ug.Nodules (3.1-5.0 mg) microdissected from C3H mice 62 weeksafter initiation of NNK, had enzyme activity comparable (370± 63 dpm/,ug) with that observed in nodules from A/J mice.DNA-MTase Expression in Hyperplastic Lesions and Tu-

mors. The increase in DNA-MTase activity detected in lesions

Table 3. Effect of NNK treatment on DNA methylation levels intype II cells from A/J and C3H mice

DNA methylation level

Endogenous NNK-treated

Mouse dpm/ng of % dpm/ng of %strain DNA control DNA control

A/J 26,376 ± 2318 100 + 9 14,188 + 2426* 185 ± 29*C3H 31,877 ± 7240 100 ± 22 26,301 + 2404 120 ± 10

Mice were sacrificed 7 days after the last NNK treatment. Values aremean ± SEM from three to four different sets of cell isolations pertime point. DNA methylation levels are inversely related to the countsobtained with the Sss I methylase-based assay; that is, a decrease incounts indicates an increase in methylation. The expression of DNAmethylation levels as a percentage of control was calculated. *, P < 0.01when compared with endogenous methylation levels from A/J mice.

Table 4. Tumor development and multiplicity in A/J and C3Hmice following treatment with NNK

No. of surface

Weeks after lesions per mouse

initiation of NNK lungtreatment A/J C3H

20 28 4 028 31 3 036 48 + 4 ND44 27 + 2 ND52 36+ 4 10 +260 ND 13 + 368 ND 12 + 1

Mean ± SEM from 10 mousedetermined.

lungs per time point. ND, not

microdissected during tumor progression in A/J mice indicatesthat changes in expression of this gene should be detectable inneoplastic cells and possibly in preneoplastic cells. A nonra-dioactive RNA in situ hybridization assay was developed usinga biotinylated oligonucleotide probe complementary to theDNA-MTase gene. Focal alveolar epithelial hyperplasia, ad-enomas, and adenocarcinomas observed in the lung sectionsfrom A/J mice sacrificed 52 weeks after initiation of NNKtreatment were examined for expression of DNA-MTase.Expression was detected by in situ hybridization with theantisense probe at all stages of tumor progression. Epithelialcells in these proliferative lesions showed cytoplasmic stainingthat varied from pale, diffuse pink to intense red cytoplasmicstaining (Fig. 2 A, D, E, and F). In addition, increasedDNA-MTase expression was detected in some hypertrophicalveolar type II cells that were in pulmonary parenchymaperipheral to lesions (Fig. 2D). DNA-MTase expression was

400

** -

300 -

100-

_*

0.30.5 0.1.0 1.1-1.5 1.3.0 3.1-5.0Lesion Size (mg)

FIG. 1. DNA-MTase activity in A/J mouse lung tumors. Tumorswere microdissected from the right cardiac or apical lobe and assayedfor DNA-MTase activity. Nodules with the smaller mass (0.3-0.5 mg)are predominantly hyperplasias and small adenomas; adenomas andcarcinomas comprise nodules of intermediate size (0.6-1.5 mg), andcarcinomas are most prevalent in nodules of 3.1-5.0 mg. Values aremeans ± SEM from 10 nodules per size group. *, P < 0.05 whencompared with lesions 0.3-0.5 mg; **, P < 0.01 when compared withlesions 0.3-1.5 mg.

Genetics: Belinsky et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

not evident in stromal cells and alveolar macrophages. Nocytoplasmic staining was present in the proliferative lesions orhypertrophic alveolar type II cells in serial sections incubatedwith the sense probe (Fig. 2B), or in the absence of probe.Sections treated with RNase prior to hybridization with theantisense probe also lacked cytoplasmic staining (Fig. 2C).

DISCUSSIONThe results of this investigation demonstrate for the first timethat modulation ofDNA-MTase activity can be cell-specific, is

segregated with susceptibility, and may also represent an earlyevent in neoplastic evolution. The 3-fold increase in DNA-MTase activity in alveolar type II cells from NNK-treated A/Jmice was similar to that observed in a study of NIH 3T3 cellstransfected with an exogenous mouse DNA-MTase gene (3).In both studies, changes in DNA-MTase activity were reflectedby profound changes in DNA methylation levels that wereassociated with altered cellular phenotype, either in the formof morphological transformation of NIH 3T3 cells or theinduction of alveolar type II cell-derived tumors in the A/Jmouse lung. These results suggest that the marked increase in

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*F......· ....~ ......

* 'e'~ rI *j.~i~is"J~~

FIG. 2. DNA-MTase expression in a hyperplastic lesion and tumors from A/J mice. (A) In situ mRNA hybridization with an antisenseDNA-MTase oligonucleotide probe produced specific staining of the papillary adenocarcinoma (upper left; bright pink to red stain), and severalof the hypertrophic alveolar type II cells in the adjacent lung were intensely stained red. (B) DNA-MTase control sense probe applied to a serialsection did not produce staining of the adenocarcinoma or the adjacent lung tissue. (C) The signal was obliterated by RNase treatment of the tissuein a serial section prior to hybridization with the antisense probe. (D) Higher magnification ofA illustrating the specificity of the staining reaction.Note lack of staining of the stroma and macrophages and the intense stain of selected type II cells (arrowheads). (E) Hybridization with the antisenseDNA-MTase oligonucleotide probe produced specific pink to red staining of many of the type II cells in the focus of alveolar epithelial hyperplasia.(F) Heterogeneous intensity of staining of epithelial cells in a pulmonary adenoma. Note that the stromal cells and lymphocytes are not stained.

4048 Genetics: Belinsky et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 4049

DNA-MTase activity detected in alveolar type II cells aftercarcinogen treatment could be a major factor contributing tothe high susceptibility for chemical-induced neoplasia associ-ated with the A/J mouse strain.The increase in DNA-MTase activity detected in lung

tumors from the more resistant C3H mice indicates thatalterations in the activity of this enzyme are not restricted toA/J mice. Most likely these changes occur within a populationof alveolar type II cells in C3H mice too small to produce adetectable change in DNA-MTase levels immediately follow-ing carcinogen exposure. This segregation of DNA-MTaseactivity with respect to lung tumor susceptibility followingNNK treatment parallels the marked differences in tumorlatency and multiplicity exhibited by these two mouse strainsand has been linked to a difference in mutation rate and/orexpression of the K-ras gene (23-25). The increase in DNA-MTase activity may also be linked to the ras signal transductionpathway. Mutation and/or overexpression of the K-ras genestimulates myc and the jun/fos complex to increase AP-1transcription factors (for a review, see ref. 26), which couldmodulate DNA-MTase gene transcription through binding toAP-1 sites present in the 5' regulatory region of this gene (27).In support of this hypothesis, DNA-MTase promoter activityis induced in P19 cells following transient transfection of c-junor H-ras (28). In addition, decreasing ras activity throughforced expression of GAP120 in the murine adrenocorticaltumor cell line Y1 has resulted in DNA demethylation andabrogation of the transformed phenotype (29).The increase in DNA-MTase activity during tumor progres-

sion in this murine lung cancer model parallels that observedin human colon cancer (2). The effect of modulating DNA-MTase activity on the development of intestinal neoplasia hasbeen examined in Min mice by Laird et al. (4). Min mice, whichcontain a mutation of the adenomatosis polyposis coli gene,develop multiple intestinal adenomas within the first fewmonths of life. DNA-MTase activity and the development ofadenomas were both reduced 50% in an F1 mouse generatedby crossing the C57BL/6 APCMin/+ mouse with a 129/SvDnmts/+ mouse that contains one mutationally inactive alleleof the DNA-MTase gene. Adenoma formation was virtuallyabolished in F, mice following treatment with the demethyl-ating agent 5-azadeoxycytidine, demonstrating a critical rolefor DNA-MTase in the development of intestinal adenomas.Studies by MacLeod and Szyf (30) also showed that transfec-tion of the Y1 cell line with an antisense construct of theDNA-MTase cDNA causesDNA demethylation and decreasestumorigenicity by this cell line in syngeneic mice. With respectto lung carcinogenesis, previous studies in a Syrian Goldenhamster model for bronchogenic cancer (31) examined theeffect of 5-azacytidine on lung cancer development. In animalsthat received 5-azacytidine 3-5 days after carcinogen exposure,tumors were significantly smaller than in animals receivingtherapy late in cancer development or no treatment, suggestingthat 5-azacytidine treatment inhibited the promotional phaseof lung cancer development in this model.

Increased DNA-MTase activity could affect tumor progres-sion through several mechanisms. Regional increases in meth-ylation in normally unmethylated cytosine-rich areas havebeen associated with alterations in chromatin structure thatmight generate DNA instability in the form of allelic loss (32,33) and lead to inactivation of multiple tumor suppressor genes(5-10). DNA methylation could also result in changes in DNAsequences, because methylated cytosine is a highly mutablebase in the eukaryotic genome (11). Alternatively, increasedexpression of the DNA-MTase gene may also cause highmutation rates at CpG sites by direct enzymatic deaminationof cytosine (12). The T:G mismatches created by this processare repaired at rates 6000-fold less than U:G matches (34).Thus, increased DNA-MTase activity in conjunction with

differences in repair efficiencies most likely underlies the C toT transitions detected at CpG sites.

Regardless of the mechanisms responsible for gene dysfunc-tion that underlie increased DNA-MTase, our findings havegreat relevance for improving the early diagnosis, prevention,and treatment of lung cancer. Genetic abnormalities arepresent throughout the respiratory tract of smokers stemmingfrom the diffuse, chronic exposure of the lungs to carcinogensand promoters contained in tobacco smoke. For example,mutations in the p53 gene and loss of heterozygosity atchromosome 3p have been reported in bronchial epitheliumexhibiting mild and severe dysplasia recovered from areas inclose proximity to the primary tumor mass (35). Recent studiesin our laboratory (R. Crowell, unpublished studies) and others(36) have detected chromosome abnormalities in normalbronchial epithelium indicating that premalignant cells may bedispersed throughout the lung. The increase in DNA-MTaseexpression detected in hypertrophic alveolar type II cells in thecurrent investigation supports the hypothesis that elevatedlevels of expression of this gene could be a biomarker forpremalignancy. If alterations in the DNA-MTase pathwayprovide premalignant cells with a selective growth advantage,then determining whether bronchial cells or dysplastic lesionsalso overexpress DNA-MTase may better define lung cancerrisk. Furthermore, the modulation of genes through DNAmethylation may substantially impact the development of lungcancer. Affecting the pathway(s) responsible for gene activa-tion or silencing could be an effective mechanism for prevent-ing, slowing, or eliminating tumor growth. In this regard,5-azadeoxycytidine has been given in combination with am-sacrine to patients with acute myeloid leukemia (37) who hadrelapsed following a high-dose cytosine arabinoside/amsacrine regimen. Eight of 11 patients obtained a completeremission with a median remission duration of 7 months. Theseencouraging results have led to the initiation of clinical trialsto examine the ability of 5-azadeoxycytidine to affect thegrowth of acute myeloid leukemia and the myelodysplasticsyndrome (38). The detection of premalignant lung diseasethrough increased DNA-MTase expression and the possibilityof blocking deleterious effects of this change with specificinhibitors can offer new intervention strategies for lung cancertreatment directed at modulating gene expression by affectingDNA-MTase activity.We thank Ximena Galarza-Johnston, Heidi Harms, Kelly Avila, and

Richard Jaramillo for excellent technical assistance. This research wassupported by the Office of Health and Environmental Research, U.S.Department of Energy under Contract DE-AC04-76EV01013 and bythe Lung Specialized Program of Research Excellence Grant CA-58184-01 in facilities fully accredited by the American Association forAccreditation of Laboratory Animal Care.

1. Bestor, T., Laudano, A., Mattaliano, R. & Ingram, V. (1988) J.Mol. Biol. 203, 971-983.

2. Issa, J.-P. J., Vertino, P. M., Wu, J., Sazawal, S., Celano, P.,Nelkin, B. D., Hamilton, S. R. & Baylin, S. B. (1993) J. Natl.Cancer Inst. 85, 1235-1240.

3. Wu, J., Issa, J.-P., Herman, J., Bassett, D. E., Nelkin, B. D. &Baylin, S. B. (1993) Proc. Natl. Acad. Sci. USA 90, 8891-8895.

4. Laird, P. W., Jackson-Grusby, L., Fazeli, A., Dickinson, S. L.,Jung, W. E., Li, E., Weinberg, R. A. & Jaenisch, R. (1995) Cell81, 197-205.

5. Herman, J. G., Latif, F., Yongkai, W., Lerman, M., Zbar, B., Liu,S., Samid, D., Dah-Shuhn, R., Gnarra, J. R., Linehan, W. M. &Baylin, S. B. (1994) Proc. Natl. Acad. Sci. USA 91, 9700-9704.

6. Ottavian, Y. L., Issa, J.-P., Parl, F. F., Smith, H. S., Baylin, S. B.& Davidson, N. E. (1994) Cancer Res. 54, 2552-2555.

7. Issa, J.-P. J., Ottaviano, Y. L., Celano, P., Hamilton, S. R., Dav-idson, N. E. & Baylin, S. B. (1994) Nat. Genet. 7, 536-540.

Genetics: Belinsky et aL.

Proc. Natl. Acad. Sci. USA 93 (1996)

8. Steenman, M. J. C., Rainier, S., Dobry, C. J., Grundy, P., Horon,I. L. & Feinberg, A. P. (1994) Nat. Genet. 8, 433-439.

9. Merlo, A., Herman, J. G., Mao, L., Lee, D. J., Gabrielson, E.,Burger, P. C., Baylin, S. B. & Sidransky, D. (1995) Nat. Med. 1,686-692.

10. Makos, M. W., Biel, M. A., Deiry, W. E., Nelkin, B. D., Issa,J.-P., Cavenee, W. K., Kuerbitz, S. J. & Baylin, S. B. (1995) Nat.Med. 6, 570-577.

11. Barker, D., Schafer, M. & White, R. (1984) Cell 36, 131-138.12. Rideout, W. M., Coetzee, G. A., Olumi, A. F. & Jones, P. A.

(1990) Science 249, 1288-1249.13. Shimkin, M. (1940). Arch. Pathol. 29, 235-255.14. Belinsky, S. A., Devereux, T. R., Foley, J. F., Maronpot, R. R. &

Anderson, M. W. (1992) Cancer Res. 52, 3164-3173.15. Malkinson, A. (1989) Toxicology 54, 241-271.16. Belinsky, S. A., Lechner, J. F. & Johnson, N. F. (1995) In Vitro

Cell. Dev. Biol. 31, 361-366.17. Foley, J. F., Anderson, M. W., Stoner, G. D., Gaul, B. W., Hard-

isty, J. F. & Ward, J. M. (1991) Exp. Lung Res. 17, 157-168.18. Adams, R. L. P., Rinaldi, A. & Seivwright, C. (1991) J. Biochem.

Biophys. Methods 22, 19-22.19. Marmur, J. A. (1961) J. Mol. Biol. 3, 208-218.20. Iezzoni, J. C., Kang, J.-H., Bucana, C. D., Reed, J. A. & Brigati,

D. J. (1993) J. Clin. Lab. Anal. 7, 247-251.21. Radinsky, R., Bucana, C. D., Ellis, L. M., Sanchez, R., Cleary,

K. R., Brigati, D. J. & Fidler, I. J (1993) Cancer Res. 53, 937-943.22. Devereux, T. R., Anderson, M.W. & Belinsky, S.A. (1991)

Carcinogenesis 12, 299-303.23. Ryan, J., Barker, P. E., Nesbitt, M. N. & Ruddle, F. H. (1987) J.

Natl. Cancer Inst. 79, 1351-1357.

24. You, M. Wang, Y., Stoner, G., You, L., Maronpot, R., Reynolds,S. H. & Anderson, M. W. (1992) Proc. Natl. Acad. Sci. USA 89,5804-5808.

25. Chen, B., Johanson, L., Wiest, J. S., Anderson, M. W. & You, M.(1994) Proc. Natl. Acad. Sci. USA 91, 1589-1593.

26. Marx, J. (1993) Science 260, 1588-1590.27. Rouleau, J., Tanigawa, G. & Szyf, M. (1992). J. Biol. Chem. 267,

7368-7377.28. Rouleau, J., MacLeod, A. R. & Szyf, M. (1995)J. Biol. Chem. 270,

1595-1601.29. MacLeod, A. R., Rouleau, J. & Szyf, M. (1995)J. Biol. Chem. 270,

11327-11337.30. MacLeod, A. R. & Szyf, M. (1995)J. Biol. Chem. 270,8037-8043.31. Hammond, W.G., Yellin, A., Gabriel, A., Paladugu, R.R.,

Azumi, N., Hill, L. R. & Benfield, J. R. (1990) Exp. Mol. Pathol.53, 34-51.

32. Antequera, F., Boyes J. & Bird, A. P. (1990) Cell 62, 503-514.33. de Bustros, A., Nelkin, B. D., Silverman, A., Ehrlich, G., Poiesz,

B. & Baylin, S. B. (1988) Proc. Natl. Acad. Sci. USA 85, 5693-5697.

34. Schmutte, C., Yang, A. S., Beart, R. W. & Jones, P. A. (1995)Cancer Res. 55, 3742-3746.

35. Sozzi, G., Miozzo, M., Donghi, R., Pilotti, S., Cariani, C. T.,Pastorino, U., Della-Porta, G. & Pierotti, M. A. (1992) CancerRes. 52, 6079-6082.

36. Pastorino, U., Sozzi, G., Miozzo, M. Tagliabue, E., Pilotti, S. &Pierotti, M. A. (1993) J. Cell Biochem. (Suppl.) 17F, 237-248.

37. Richel, D. J., Colly, L. P., Kluin-Nelemans, J. C. & Willemze, R.(1991) Br. J. Cancer 64, 144-148.

38. Pinto, A. & Zagonel, V. (1993) Leukemia 7, 51-60.

4050 Genetics: Belinsky et al.