altered chromosomal methylation patterns accompany ... · altered chromosomal methylation patterns...

[CANCER RESEARCH 53, 1684-1689, April 1. 1993|

Altered Chromosomal Methylation Patterns Accompany Oncogene-inducedTransformation of Human Bronchial Epithelial Cells1

Paula M. Ventino,2 Elisa A. Spillare, Curtis C. Harris, and Stephen B. Baylin

Oncology Center Â¡P.M. V., S. B. B.Â¡and Department of Medicine [S. B. B./, The Johns Hopkins University School of Medicine, Baltimore. Maryland 21231, and Laboratory ofHuman Carcinoxenesis Â¡E.A. S.. C. C. H.Â¡,National Cancer institute, NIH, Bethesda, Maryland 20892

ABSTRACT

Abnormal methylation of CpG island sequences on chromosomes lipand 17p, and tumor phenotype-associated differential methylation of chro

mosome 3p loci have been described in human lung tumors (S. B. Baylin,J. W. M. Hoppener, A. de Bustros, P. H. Steenbergh, C. J. M. Lips, and B.D. Nelkin, Cancer Res., 46: 2917-2922, 1986; M. Makos, B. D. Nelkin, M.

I. Lerman, F. Latif, B. Zbar, and S. B. Baylin, Proc. Nati. Acad. Sci. USA,89: 1929-1933,1992; A. de Bustros, B. D. Nelkin, A. Silverman, G. Ehrlich,B. Poiesz, and S. B. Baylin, Proc. Nati. Acad. Sci. USA, S5: 5693-5697,

1988). Using an in vitro model of lung tumor progression, we now showthat these aberrant methylation patterns occur at different stages duringcellular immortalization and oncogene-induced neoplastic transformation

of normal human bronchial epithelial cells (NHBE). The CALCI CpGisland locus on chromosome Hpl5.4 was essentially unmethylated inNHBE and simian virus 40 T-antigen immortalized NHBE (BEAS-2Bcells) but became de novo methylated in 5 of 6 BEAS-2B derived cell lines

that were transfected or infected with various oncogenes and in a spontaneously neoplastically transformed subline of BEAS-2B cells. By con

trast, an additional CpG island locus, pYN/,22. at 17pl3.3 became fullymethylated following the immortalization of NHBE and was not furtherchanged by oncogene-induced transformation. Finally, at a non-CpG island locus pYNZ86.1 on chromosome 3pl4, different tumor phenotype-

associated methylation patterns became apparent only after passage of thetumorigenic oncogene-transformed bronchial epithelial cell lines in athy-mic nude mice. Whereas cell lines derived from tumors with a non-smallcell lung carcinoma-like phenotype were significantly hypomethylated rel

ative to their parental cell lines, a cell line derived from a tumor with amore small cell lung carcinoma-like phenotype retained the methylation

status of its parental cell line. The data indicate that altered DNA methylation patterns, including the de novo methylation of normally unmethylated CpG island sequences and demethylation of nonisland sequences,arise at different stages during immortalization and oncogene-induced

neoplastic transformation of bronchial epithelial cells. These findings suggest that DNA methylation abnormalities accompany, or may play a rolein, the genetic changes that occur during lung tumor progression.

INTRODUCTION

Alterations in genomic DNA methylation levels and patterns arecommon in human cancers. Relative to normal cells, cancer cellsexhibit an overall decrease in the levels of 5-methylcytosine (1),

increased expression of the DNA methyltransferase gene (2), and aredistribution of methylation patterns. These altered patterns includethe demethylation of normally methylated regions, such as bulk chro-

matin and the bodies of certain genes, and the dv novo methylation ofnormally unmethylated areas, such as CpG "islands" (reviewed in

Refs. 3 and 4). CpG islands are C + G-rich areas of the genome

which, relative to bulk chromatin, contain a high frequency of themethylatable dinucleotide CpG (5). With the exception of CpG islandson the inactive X chromosome where methylation may play a role in

Received 12/7/92; accepted I/2HTO.The costs of publication Â»I'this article were defrayed in part by the payment of page

long-term transcriptional repression, CpG islands are unmethylated in

normal adult tissues (5). The promoters of most housekeeping andseveral tissue-specific genes lie within CpG islands, and the methy-lation-free status of these sequences is thought to be necessary, but not

sufficient, for active transcription to occur (reviewed in Ref. 5). Inappropriate methylation of CpG island sequences in tumors (4, 6-8) or

cells in culture (9, 10) may interfere with the normal expression ofgrowth-suppressing or differentiation-inducing genes. Given the cur

rent interest in gene inactivation as a primary event during tumorprogression, the identification of hypermethylated DNA regions inhuman tumors may be of particular importance.

In an ongoing effort to determine the relationship between alteredDNA methylation and tumorigenesis, we have recently identified several chromosomal loci which are hypermethylated in human tumors.CpG island areas on chromosomes lip and 17p are consistentlyhypermethylated in diverse human tumors, including lung and colon(6-8, 11). Abnormal methylation of both loci occurs early during

colon tumor progression, appearing at the benign adenoma stage(7, 11). At the 17p locus, CpG island hypermethylation precedesallelic loss (7). In addition, we have found that in SCLCs1 hyperm

ethylation occurs at two loci on chromosome 3p that are frequentlyreduced to homozygosity in this tumor type (7). These data suggestthat, in tumor cells, hypermethylation may participate in chromosomalrearrangement or loss, or may mark unstable genomic regions.

In this study, we use a well-defined in vitro model of lung carcino-

genesis to examine the timing of altered DNA methylation events inthe evolution of lung cancer. NHBE are believed to represent theprogenitor cell for lung carcinomas, and means of culturing these cellshave previously been established (12). Immortalization of NHBE withan SV40-adenovirus Eia hybrid virus has led to the establishment ofa cell line, BEAS-2B, which retains many of the properties of NHBE

such as undergoing terminal differentiation in response to serum ortransforming growth factor ÃŸ( 13, 14). The subsequent introduction ofthe oncogenes v-Ha-ra.v, v-Ki-ra.s, mutant p5.i, or the combination ofc-myc and c-ruf causes BEAS-2B to undergo tumorigenic conversion(15-18). Cell lines harboring each of these oncogenes, as well as lines

derived from the corresponding tumors, have been developed. Sincethe cell lines have a common origin (BEAS-2B cells), the directeffects of oncogene-induced transformation on methylation patterns

can be assessed. The methylation status of the bronchial epithelial celllines was determined at loci on chromosomes lip, 17p, and 3p thathave shown altered methylation patterns in established lung tumors.The data indicate that altered DNA methylation patterns, including the(te novo methylation of CpG island sequences, accompany the immortalization and oncogene-induced neoplastic transformation of

bronchial epithelial cells.

MATERIALS AND METHODS

Cell Lines. The establishmentof the cell lines used in this study hascharges. This article must therefore be hereby marked lulvenisemtni in accordance with previously been described (12, 13. 15-17). Briefly, primary bronchial epilhe-

IK U.S.C. Section 1734 solely to indicate this fact.1Supported by NIH Grants IPM) CA5HI84 and 5ROI CA43318.2 To whom requests for reprints should he addressed, at 424 N. Bond St.. Baltimore.

MD 2123I.

1The abbreviations used are: SC'I.C. small cell lung carcinoma: Nf IBP., normal human

bronchial epithelial cells; SV40. simian vims 40.

I6K4

on June 24, 2020. © 1993 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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ALTERED DNA METHYLATION IN HUMAN BRONCHIAL EP1THKI.IAL CELLS

lium and expiant cultures of NHBE were derived from autopsy specimens asdescribed (12). BEAS-2B cells are an immortalized, large T-antigen-producingderivative of NHBE that were generated by infection of NHBE with an ade-

novirus I2-SV40 hybrid virus (13). The B39-TLcell line was established froma slow-growing tumor produced after injection of passage 39 BEAS-2B cellsinto athymic nude mice.4 BEAS-2B cells infected with Kirsten murine sarcoma

virus and propagated as a mass culture are designated BVK ( 15). BVK-T11

cells were generated from an athymic nude mouse tumor produced by s.c.injection of BVK cells (15). BEAS-2B cells infected with a Zip-neoSV(X)recombinant retrovirus containing the v-Ha-ras gene were selected in G418.and resistant cells were pooled and designated BZR ( 16). BZR-T33 cells were

generated from an athymic nude mouse tumor produced by s.c. injection ofBZR cells (16). 2B-Z\p-neo, 2B-mvc. and 2B-ra/cells were produced bytransfection of BEAS-2B cells with the pZip-/ipÂ»SV(X) plasmid vector (2B-Zip-nfo) or a pZip-nioSV(X) construct containing either murine c-/mr (2B-mvc) or human c-ra/(2B-ra/) complementary DNAs (17). 2B-ra//wvr cells areBEAS-2B cells that have been transfected with both the c-mvc- and c-raf

containing pZip-neo constructs (17). These cell lines were grown as masscultures of G418-resistant cells. RMT-2 cells were generated from an athymicnude mouse tumor produced by s.c. injection of 2B-ra//mvc- cells (17). 2B-

CMV-HCi) and 2B-MT249 cells were derived from the transfection of BE-AS-2B cells with a pCMV-neo plasmid (2B-CMV-;ifo) or a pCMV-neo con

struct containing a human f>53 complementary DNA with a mutation at codon249.s Cells were selected in G418. and individual resistant colonies were

ring-cloned and expanded. These clonal cell lines are designated 2B-MT249

cl.8 and cl.10. All of the bronchial epithelial cell lines were maintained onfibronectin/collagen-coated dishes in LHC-9 medium, with the exception ofRMT-2 cells, which were grown in LHC-9 medium plus 1% fetal bovineserum. SV40-immortalized human fibroblasts were obtained from the NIA

Aging Cell Repository AG02804C (Camden. NJ).Genomic Probes. The 5 ' region of the human calcitonin (CALCI ) gene has

been described previously (6. 8. 11. 19). The probe used in this study was a0.6-kilobase BgH\-BiimH\ fragment extending from nucleotides -728 to -138of the genomic sequence. The 1.7-kilobase genomic pYNZ22 fragment was agift from Dr. Bert Vogelstein. The 3.3-kilobase genomic probe pYNZ86.1 was

obtained from the American Type Culture Collection (Rockville. MD). Isolatedprobe fragments were labeled with 12P by the random-hexamer priming

method (20).Methylation Analyses. DNA was isolated from cultured cells essentially as

described (6). DNA methylation was assessed by digestion of 10 ug DNA withthe isoschizomeric restriction enzymes Mspl. which cuts DNA at its targetsequence CCGG regardless of the methylation status of the internal cytosine.or ///Kill, which is inhibited if this cytosine is methylated. Samples were alsodigested with Hindlll. a non-methylation-sensitive enzyme, to provide anoutside flanking cut. Restrictions were performed for 16 h at 37Â°C at a

concentration of 20 units enzyme/ug DNA to ensure complete digestion. Whereindicated. 5 ug DNA was digested with the cytosine methylation-sensitiveenzyme Noil. Digested samples were electrophoresed on l"7r agarose gels,

transferred to nylon membranes (Zetaprobe, Biorad). and hybridized overnightat 60Â°C to 1-5 x 107 cpm probe according to standard procedures (21).

Membranes were washed to a final stringency of 0.1 x standard saline citrate(IX = 0.15 MNaCl, 0.015 Msodium citrate), 0.1% sodium dodecyl sulfate at65Â°Cand exposed to Kodak XAR-5 film. Blots were stripped between hybridizations by incubation at 95Â°Cin 0.1 x standard saline citrate, 0.1 "7rsodium

dodecyl sulfate for 20 min.

RESULTS

Methylation of the CALCI Locus CpG Island at Ilpl5.4. Thebronchial epithelial cell lines were first examined for methylation ofthe CpG island in the 5' end of the calcitonin (CALCI) gene located

at chromosome 1Ipl5.4. Identification of this region as a CpG islandbased on C + G content. CpG frequency, and CpG-containing rare-

4 R. R. Reddel. S. E. Salgheiti. J. C. Willey, Y. Ohnuki. Y. Ke, B. I. Gerwin. J. F.

Lechner. and C. C. Harris. Development of tumorigenicity in SV40-imrnortali/ed humanbronchial epithelial cells, submitted tor publication.

5 W. Bennett et al., unpublished results.

base restriction enzyme sites has been described ( 19). Previous studieshave indicated that Mspl sites within this region are unmethylated innormal lung tissue but are hypermethylated in lung tumor tissue andestablished lung cancer cell lines (6, 8). The methylation status ofMspl sites in this region was assessed by Southern blot analysis ofMspl- or W/xiII-digested DNA using a probe extending from nucleotides -728 to -138 of the CALCI gene promoter (see Fig. IA).

Comparison of DNA from normal lung tissue (not shown) and expiantcultures of NHBE (Fig. IÃŸ)showed similar methylation patterns.DNA from both cell types had a minor 1.1-kilobase band in ///will-digested samples in addition to the expected 0.5- and 0.6-kilobase

bands present in Mspl digests, indicating the presence of partial methylation at a single Mspl site in a small fraction of cells (see Fig. 1/4,site 12). Thus, with the exception of partial methylation of site 12, theCALCI CpG island was essentially unmethylated in both normal lungtissue and NHBE cells.

To determine the effects of SV40 T-antigen-induced immortaliza

tion and subsequent oncogenic transformation on methylation at theCALCI CpG island. BEAS-2B cells and the oncogene-transformedBEAS-2B derivatives were also analyzed. The Hpall pattern of BE-AS-2B cells also indicated methylation of site 12 (Fig. IÃŸ).Compared

to NHBE, the degree of methylation at this site was increased, asevidenced by the increase in intensity of the 1.1-kilobase band relativeto the 0.5- andO.6-kiloba.se bands (Fig. IÃŸ).Methylation of additional

sites was not detected, indicating that immortalization of NHBE withSV40 T-antigen resulted in an increased fraction of cells with methylation of site 12 only. The subsequent introduction of various onco-genes into BEAS-2B cells caused a further increase in methylation of

A.

Mspl /Hpall1

2,3,4.3 6I III1H31.8

kbsites7

8910.111II 1i

probt12

13114215,16 17II 1+

1.8kb

0.5 kb 0.6 KbMspl Fragments

B.

S!â€ž¿�

m aa <X UJH M z m

! i i 11m m m m Z

tr Å’ * *N N > >tu ce tu co

â€¢¿�2Z

oÃm

8.0 kb - Â«.

i0.6 kb ,_0.5 kb â€¢¿�" Ã•.

Fig. l. Methylution of the CALCI CpG island at I Ipl5.4 in human bronchial epithelialcells. A, genomic map of the 5' region of the human CALCI gene at I lpI5.4 as extrap

olated from Broad et ni. ( I9). Msp\/HpaU sites (numbered 1-I7) and the relevant HinJ\\\site (H3) are indicated. A second HindlU site occurs 8 kilobases downstream of H3 (notshown). Filled boxes, exons I and 2. Distances (in kilobases) are indicated beneath theline, with respect to the transcription start site at the beginning of exon I. Stippled box,sequences covered by the probe used for Southern analysis. Also shown are the 0.5- and0.6-kilobase bands generated by Msp\ digestion and Southern analysis. B, methylationanalysis of the bronchial epithelial cell lines. Southern analysis of DNA samples from thecell line indicated above each lane digested with either HinJ\\\ (H), Hind\\\ + Msp\ (M),or Hintl\\l + HpaU (all others). Hintftli and Hinulil + Mxp\ patterns were the same forall cell lines. Restricted DNAs were electrophoresed on l*/Ãagarose gels, transferred to

nylon fillers, and hybridi/ed to the probe shown in A.

1685



ALTERED DNA METHYLATION IN HUMAN BRONCHIAL EPITHELIAL CELLS

the CALCI CpG island which involved not only site 12 but severaladditional Hpall sites (Fig. IÃŸ).This was suggested by the relativeloss of the 0.5- and 0.6-kilobase bands and the appearance of bands at0.7, 1.1, 1.3. 1.6. 2.0, and 2.2 kilobases in the //pall-digested samples.

The extent and exact pattern of hypermethylation varied with thespecific oncogene used. Whereas the 2B-mvc and 2B-ra/ cell lineswere rather modestly hypermethylated, the 2B-raflm\c, BZR, BVK,and 2B-MT249 lines were more markedly hypermethylated. Interestingly, the least methylated of the oncogene-containing cell lines (2B-raf and 2B-mvc) are also nontumorigenic in nude mice, whereas themore extensively methylated cell lines (2B-raflmyc. BVK, and BZR)are tumorigenic (15-17).

To address whether the genomic methylation changes observedwere due to the presence of the oncogene or occurred during establishment of these cell lines, two vector-only control cell lines werealso tested. These included the 2B-zip-/ie(; and the 2B-CMV-/ieY>celllines, which were derived from the stable transfection of BEAS-2Bcells with either the pZip-neo vector (control cell line for the 2B-mvc,2B-mf, and 2B-raf/myc cells) or the pCMV-nea vector (control for the2B-MT249 cells), respectively. The pattern and extent of methylation

at the CALCI CpG island in these cell types was similar to that of theparent BEAS-2B cells (Fig. IÃŸ).Interclonal variation in methylation

patterns within a cell population is also known to occur (22). Themajority of cell lines used in this study were established as masscultures (i.e., nonclonal), and it can be assumed that the methylationpatterns shown here are representative of the cell population. The2B-MT249 cells, however, were established from individual G418-resistant colonies. For this reason, two different 2B-MT249 clonal cell

lines were examined (Fig. IÃŸ).Although the exact methylation patternof the CALCI CpG island did vary between the two clones, both weremarkedly hypermethylated.

To determine the effects of in vivo tumor growth on CALCI CpGisland methylation. cell lines derived from nude mouse tumors produced by the 2B-raflm\c, BVK, and BZR cell lines (RMT-2, BVK-Tl 1, and BZR-T33, respectively) and a cell line derived from a rarespontaneous tumor of BEAS-2B cells (B39-TL) were also analyzed(Fig. IÃŸ).Although some of the tumor-derived lines were more extensively methylated than their respective parent lines (BVK-T11versus BVK and B39-TL versus BEAS-2B), and others were lessmethylated (BZR-T33 versus BZR and RMT versus 2B-raf/myc), it isclear that all of the tumor-derived cell lines were abnormally methy

lated at the CALCI locus.Methylation of the pYNZ22 Locus at 17pl3.3. We next studied

the methylation status of a CG-rich locus on chromosome 17pl3.3.

The probe, pYNZ22, recognizes a highly polymorphic, variable number tandem repeat region that is frequently used to assess the allelicstatus of chromosome 17p (23). This region contains 5 Noti restrictionsites within ~8 kilobases of DNA (Fig. 2A) (24). Noti is a methyla-tion-sensitive, "rare base" restriction enzyme recognizing the 8-basepair sequence 5'-GCGGCCGC-3'. It is estimated that 89% of ge

nomic Noti sites occur within CpG islands (25). The region surrounding the pYNZ22 locus is therefore believed to contain one or moreCpG islands. We have previously found this locus to be extensivelyhypermethylated in lung and colon tumors (7).

Noti digestion and Southern analysis of the pYNZ22 region recognized polymorphic bands in the 4.6-5.0-kilobase range in DNA from

normal lung tissue (7) and immortalized human lung fibroblasts (Fig.2ÃŸ.Lane I ). Expiant cultures of NHBE. however, showed a partiallymethylated pattern with the appearance of bands at ~8.0 kilobasesand >23 kilobases in addition to the normal 4.6-kilobase band(s),

indicating that methylation had occurred at one or more of Noti sites2-6 (Fig. 2B, Lane 3). DNA from the SV40 T-antigen-immortalized

A.

3 kb â€¢¿�

B.

>23 kb -

8.0 kb -

4.6 kb -

l i f"z LU en i

00 < 0Â»o X tu n~- z m m

tÂ»

o ~ ^C O) O)

022

Fig. 2. Methylation of the pYNZ22 region at 17pl3.3 in human bronchia! epithelialcells. A. genomic map of the region surrounding the pYNZ22 probe adapted from Ref. 24(not to scale). /VÂ»/lsites are numbered 1-8. Approximate distances between Noi\ sites areindicated, except within the 3-kilohase region, where they are not known. Sti/ipled hox,sequences covered by the 1.7-kilobase pYNZ22 probe used for Southern analysis, fl.methylation analysis of DNA samples from SV40-transformed fibroblasts iFihnt), primary uncultured bronchial epithelial cells ( 1Â°NHBE), or the indicated bronchial epithelial

cell line. DNA samples were digested with Null, electrophoresed on n agarose gels,transferred to nylon filters, and hybridized to the probe pYNZ22 (shown in A}. Slightdifferences in migration of the 8.0-kilobase bands between the primary and cultured

NHBE are due to differences in electrophoresis conditions rather than true differences inband size.

BEAS-2B cells was completely methylated at these Noti sites, causing

a shift in the band size to the unresolved portion of the gel (>23kilobases) (Fig. 2ÃŸ).In general, this maximal hypermethylation wasretained in the oncogene-transformed BEAS-2B cell lines and thetumor-derived cell lines with only a slight loss of methylation in theBZR-T33 and BVK-T11 cell lines, as evidenced by the presence of a

light band at 4.6 kilobases (Fig. 2ÃŸ).The fact that NHBE cells were partially methylated at Noti sites in

the pYNZ22 region raised the question of whether methylation of thisregion was either induced by cell culture or preexisted in the primarybronchial epithelium. We examined the methylation status of normalbronchial epithelium after separation from the underlying stroma andprior to any cell culture. As shown in Fig. 2B. primary epithelial cellswere also partially methylated at the pYNZ22 locus, albeit to a somewhat lesser extent than the NHBE cells (Fig. 2ÃŸ:compare Lanes 2 and3). The data suggest that partial methylation of Noti sites in thepYNZ22 locus preexists in normal bronchial epithelium, that cultureof NHBE can further increase this methylation, and that extensivehypermethylation of this region occurs during immortalization ofNHBE cells.

Methylation of the pYNZ86.1 Locus on Chromosome 3p. Thefinal studies involved the D3S30 locus on chromosome 3pl3-14

which is recognized by the pYNZ86.1 probe. This locus is consistently hypermethylated in human SCLC cell lines, but is most oftenhypomethylated in non-SCLC and colon tumors (7). The relationship

between the pYNZ86.1 probe and any CpG islands or genes is notknown, but the probe is not believed to lie directly within a CpG islandsince the locus is methylated at some Hpcill sites in normal lung andcolon tissue (7).

1686



ALTERED DNA METHY1.ATION IN HUMAN BRONCHIA!. EPITHELIAL CELLS

The oncogene-transformed BEAS-2B cell lines that produce tumors

represent, to a certain extent, the different phenotypes of lung cancer.The phenotype of tumors in athymic nude mice giving rise to theB39-TL. BZR-T33, and BVK-T11 cell lines have been classified ascystic with squamous components.4 undifferentiated carcinomas (16),

and poorly differentiated adenocarcinomas (15), respectively. Theselines can therefore be regarded as non-SCLC phenotypes. None of thecell lines manifest the full features of SCLC, but cells of the 2B-ra/7mvc and RMT-2 lineage show some markers of the neuroendocrine

differentiation characteristic of SCLC such as dense core granules,neuron-specific enolase expression, and a keratin expression patternconsistent with variant SCLC cell lines (26). We examined the meth-ylation status of the pYNZ86.1 locus in the tumor-derived cell linesB39-TL, BZR-T33. BVK-T11. and RMT-2.

DNA from NHBE and BEAS-2B cells was methylated at Hpall

sites within the pYNZ86.1 locus, giving a pattern that was similar tothat described for normal lung tissue (Fig. 3). We found little difference in the methylation patterns between BEAS-2B cells and theoncogene-transformed cell lines 2B-ra/, 2B-/nvr, 2B-raf/myc, BVK,BZR. and 2B-MT249 (Fig. 3 and data not shown). The most signif

icant methylation changes at this locus were noted in DNA from thecell lines derived from athymic nude mouse tumors. The B39-TL.BZR-T33. and BVK-T11 cell lines were extensively hypomethylatedrelative to their respective parental cell lines (BEAS-2B, BZR, andBVK) (Fig. 3). By contrast, RMT-2 cells retained most of the methylation present in its parental cell line, the 2B-raf/myc cells (Fig. 3).

There appeared, therefore, to be a distinction in methylation patternsat this locus between the cells of the SCLC and non-SCLC phenotypes

that was similar to that seen in established lung cancer cell lines.

DISCUSSION

The abnormal methylation of CpG island sequences on chromosomes lip and 17p and tumor phenotype-associated differential me

thylation of chromosome 3p loci have been described in human lungtumors (6-8). In this study, we used a well-defined bronchial epithelial

cell system (a) to determine whether several methylation abnormalities seen in fresh lung tumors and established lung cancer cell linescan be mimicked in an in vitro lung carcinogenesis model; and (b) toexamine the evolution of these aberrant methylation patterns as afunction of immortalization and oncogene-induced neoplastic trans

formation of bronchial epithelial cells.The data indicate that de novo methylation of normally unmethy-

lated CpG island sequences and phenotype-associated demethylation

H M

mCÂ« _l

CO I< O111 nco m

o>>

m"

lE (EN NDÃ» CD

> >OÃ™OÃ™

5.0 kb -

2.7 kb -

1.8 kb -

0.7 kb -â€¢¿�

Fig. 3. Methylation of the pYNZ86.l region at 3pl3-l4 in human bronchial epithelialcells. Southern analysis of DNA samples from the cell line indicated above each lanedigested with either Hindlll (H). Hind\ll + Mspl (M), or Hindlll + Hpall (all others).Hindlll and Htndlll + Msp\ patterns were the same for all cell lines. Restricted DNAswere electrophoresed on l<7ragarose gels, transferred to nylon filters, and hybridized tothe 3.3-kilobase pYNZ86.1 probe.

of nonisland sequences accompany immortalization and oncogene-

induced transformation of bronchial epithelial cells. Furthermore, ateach locus tested, altered methylation occurs at a different stageduring this progression. At the CALCI gene-associated CpG island on

chromosome I Ip, hypermethylation accompanied neoplastic transformation of bronchial epithelial cells. This was suggested by severalobservations. First, the CALCI CpG island was essentially unmethy-lated in NHBE and SV40 T-antigen-immortalized BEAS-2B cells butbecame de novo methylated in the oncogene-transformed cell lines.

This abnormal methylation appeared to be associated with the expression of exogenous oncogenes rather than a secondary consequence oftransfection or drug selection since neither 2B-Zip-/!tY>nor 2B-CMV-

neo cells were substantially methylated. Second, hypermethylation ofthis locus was observed in a spontaneously transformed derivative ofBEAS-2B cells (B39-TL). Finally, there appeared to be a correlationbetween the extent of CALCI CpG island methylation and tumorige-nicity among the oncogene-transformed cell lines. The tumorigeniccell lines BZR, BVK, 2B-raflmyc were more extensively methylatedthan the nontumorigenic (2B-ra/, 2B-myc) cell lines.

In contrast to the findings on lip, the I7p locus pYNZ22 becamefully methylated following SV40 T-antigen-induced immortalizationof NHBE cells and was not further changed by oncogene-induced

neoplastic transformation. This suggests that de novo methylation ofthe pYNZ22 locus is an early event in bronchial epithelial cell carcinogenesis that is associated with immortalization. The apparentsensitivity to hypermethylation is likely related to the finding thatprimary and cultured NHBE cells are partially methylated at thislocus. Several studies have suggested that once partial methylation ofCpG island DNA occurs, it may predispose a region to further methylation. Spreading of methylation within a CpG island has been observed following transfection of an in vitro methylated substrate intomammalian cells (27). Similarly, a single site in the MyoDl gene-

associated CpG island that is partially methylated in normal mousetissues may act as a nucleus from which methylation spreads uponcellular immortalization (9). Indeed, studies on the pYNZ22 region infresh colon tumors have shown that partial methylation in benignadenomas becomes more extensive after their progression to malignant carcinoma (7).

The fact that primary NHBE cells were partially methylated at Nonsites in the pYNZ22 region raises several questions. The relativeabundance of Noti and other CpG-recognizing rare base restriction

enzyme sites around the pYNZ22 locus indicate the presence of oneor more CpG islands (28). As expected for a CpG island, previousstudies had indicated that the pYNZ22 region was unmethylated inseveral normal tissues, including whole lung (7, 28). However, whenwe looked at the methylation of this locus in DNA from primarybronchial epithelium that had been separated from the underlyingstroma, it was partially methylated (Fig. 2B). The apparent specificityof partial methylation at the pYNZ22 locus for bronchial epithelium isemphasized by the finding that DNA from neither bronchial stromanor isolated primary colonie epithelium is methylated at this locus.6

Studies addressing the possibilities that partial methylation of thislocus is induced in these cells by environmental exposure or that itmay be specifically related to the differentiation of bronchial epithelium are under way.

The apparent sensitivity, and specificity, of bronchial epithelium tohypermethylation of pYNZ22 locus on 17p is also of particular interest in light of the recent findings suggesting that chromosome 17pl3deletions occur early during lung tumorigenesis, appearing in precan-cerous bronchial lesions (29). Methylation of the pYNZ22 locus pre-

' P. M. Venino and S. B. Baylin. unpublished results.

1687



ALTERED DNA METHYLATION IN HUMAN BRONCHIA!. EPITHELIAL CELLS

cedes 17p allelic loss during colon tumor progression (7), suggestingthat abnormal hypermethylation may at least mark, if not contributeto, genomic instability in this region. Allelic losses of 17p and thepYNZ22 locus have been described for a number of tumor types,including lung, brain, breast, colon, and bladder cancers (30-35). It

has been suggested that a tumor suppressor gene, distinct from the p53locus at 17pl3.1, may reside at 17pl3.3, in the vicinity of pYNZ22(35). Further study on the relationship between altered methylation,17p allelic losses, and p53 mutations in fresh lung tumors shouldclarify the role of each of these changes in lung tumorigenesis.

Studies of the 3p locus, pYNZ86.1, involve a type of methylationdifferent from that on 1Ip and 17p discussed above. This region doesnot contain a known CpG island and is methylated at some Hpall sitesin normal lung tissue (7). Previous studies indicate that methylationchanges at this locus may be associated with the major phenotypes oflung cancer. Established SCLC cell lines tend to be either hyperme-

thylated in this region or to retain the methylation levels of normallung tissue, whereas DNA from non-SCLC cell lines is most often

markedly hypomethylated (7). Consistent with this, we find that in theBEAS-2B model system the cell lines that are derived from tumorswith a non-small cell phenotype [B39-TL,4 BVK-T33 (15), and BZR-

Tll (16)] are significantly hypomethylated at this locus. Similarly,RMT-2 cells, which show some markers of the neuroendocrine dif

ferentiation characteristic of the SCLC phenotype ( 17, 26), retain themethylation levels of the BEAS-2B and the oncogene-containing pa

rental cell lines. This differential methylation became apparent onlyafter passage of the tumorigenic oncogene-transformed cells in athy-

mic nude mice, indicating that they occur as a rather late event inbronchial epithelial cell transformation. The data obtained in the BE-AS-2B model system thus provide evidence that the phenotypic traits

induced by specific oncogenes or combinations of oncogenes may belinked to the altered DNA methylation patterns we have observed inhuman lung cancers.

The results obtained for chromosomes lip and 3p in the B39-TLcells are particularly intriguing. Unlike the oncogene-transformed,tumor-derived cell lines, B39-TL cells were derived from a rare spontaneous tumor of passage 39 BEAS-2B cells.4 To date, the only

genetic difference found between B39-TL cells and BEAS-2B cells isallelic loss on chromosome 3p.4 Chromosome 3p losses are common

in human lung tumors, occurring in virtually all SCLCs and a significant proportion of non-SCLCs (e.g., Refs. 36-39). The data suggestthat CpG island hypermethylation on chromosome lip and hypome-

thylation of the nonisland locus on 3p are additional genomic changesthat accompany spontaneous transformation in bronchial epithelialcells.

In summary, the results indicate that DNA methylation changespreviously reported to occur in both fresh lung tumors and establishedlung cancer cell lines arise at various stages of tumor progression inhuman bronchial epithelium. Some of these changes appear to be quiteearly, such as hypermethylation of the CpG island on chromosome17p, while CpG island methylation on 1Ip and phenotype-associated

demethylation of a nonisland locus on chromosome 3p may occurlater. Chromosomes 17p, lip, and 3p harbor known and/or suspectedtumor suppressor genes and, as suggested by studies of the Rb gene inretinoblastomas (40), the altered methylation patterns described couldbe one mechanism by which these genes are inactivated. Hypermethylation of CpG islands may be particularly important since in vitromethylation of CpG island-containing promoter sequences is suffi

cient to inhibit transcription (5, 41). Recent studies suggest that denovo methylation of CpG island sequences and the silencing of tissue-

specific genes or genes which induce terminal differentiation mayplay a critical role in cellular immortalization and transformation

(9, 10). For these reasons, it will be of great interest to determine thetiming of altered DNA methylation events at genetic loci which undergo allelic loss and gene inactivation during progressive stages oflung cancer.

ACKNOWLEDGMENTS

The authors wish to thank Dr. Edward Gabrielson for the fresh bronchialepithelial tissue, Drs. Diane Krause and Robert Casero for their critical reviewof the manuscript, and Sandra Lund for her secretarial assistance.

REFERENCES

I.

10.

14.

15.

16.

18.

19.

20.

21.

22.

Feinberg. A. P.. Gehrkc, C. W.. Kuo. K. C, and Ehrlich, M. Reduced genomic5-methylcytosine contenÃin human colonie neoplasia. Cancer Res.. 48: 1159-1161,

1988.EI-Deiry. W. S.. Nelkin, B. D.. Celano. P.. Yen. R-W. C, Falco, J. P.. Hamilton, S. R.,

and Baylin. S. B. High expression of the DNA methyltransferase gene characterizeshuman neoplastic cells and progression stages of colon cancer. Proc. Nati. Acad. Sci.USA. 88: 3470-3474. 1991.Jones. P. A., and Buckley, J. D. The role of DNA methylation in cancer. Adv. CancerRes., 54: 1-23, 1990.Baylin, S. B., Makos, M., Wu, J., Yen. R-W. C., de Bustros, A., Venino. P.. and Nelkin.B. D. Abnormal patterns of DNA methylation in human neoplasia: potential consequences for tumor progression. Cancer Cells (Cold Spring Harbor), .*: 383-390. 1991.Bird. A. P. CpG-rich islands and the function of DNA methylation. Nature (Lond.).Â¡21:209-213, 1986.

Baylin. S. B., Hoppener. J. W. M.. de Bustros, A., Steenbergh, P. H.. Lips, C. J. M.,and Nelkin. B. D. DNA methylation patterns of the calcitonin gene in human lungcancers and lymphomas. Cancer Res., 46: 2917-2922, 1986.Makos, M.. Nelkin. B. D.. Lerman, M. I., Latif. F., Zbar, B.. and Baylin. S. B. Distincthypermethylation patterns occur at altered chromosome loci in human lung and coloncancer. Proc. Nati. Acad. Sci. USA, 89: 1929-1933, 1992.de Bustros. A., Nelkin, B. D., Silverman. A., Ehrlich. G.. Poiesz. B., and Baylin, S.B. The short arm of chromosome 11 is a "hot spot" for hypermethylation in human

neoplasia. Proc. Nati. Acad. Sci. USA. 85: 5693-5697, 1988.Jones, P. A.. Wolkowicz, M. J., Rideout, W. M.. Gonzales, F. A., Marziasz, C. M.,Coetzee. G. A., and Tapscott, S. J. De nom methylation of the MyoDI CpG islandduring the establishment of immortal cell lines. Proc. Nati. Acad. Sci. USA. 87:6117-6121, 1990.

Antequera, F.. Boyes, J., and Bird. A. High levels of de novo methylation and alteredchromatin structure at CpG islands in cell lines. Cell, 62: 503-514, 1990.Silverman. A. L.. Park, J-G.. Hamilton, S. R.. Gazdar. A. F., Luk. G. D., and Baylin.S. B. Abnormal methylation of the calcitonin gene in human colonie neoplasms.Cancer Res.. 49: 3468-3473, 1989.Lechner. J. F., and LaVeck. M. A. A serum-free method for culturing normal humanbronchial epithelial cells at clonal density. J. Tissue Culture Methods, 9: 43^t8, 1985.Reddel, R. R., Ke, Y.. Gerwin, B. I., McMenamin, M. G., Lechner, J. F., Su, R. T.,Brash, D. E., Park, J-B., Rhim, J. S.. and Harris, C. C. Transformation of humanbronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus,or transfection via strontium phosphate coprecipitation with a plasmid containingSV40 early region genes. Cancer Res., 48: 1904-1909, 1988.Ke, Y., Reddel. R. R., Gerwin, B. !.. Miyashita. M., McMenamin, M.. Lechner, J. F.,and Harris, C. C. Human bronchial epithelial cells with integrated SV40 virus Tantigen genes retain the ability to undergo squamous differentiation. Differentiation,38: 60-66, 1988.Amstad, P., Reddel, R. R.. Pfeifer. A., Malan-Shibley. L., Mark, G. E., and Harris, C.C. Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey rat. Mol. Carcinog.. /: 151-160, 1988.Reddel. R. R., Kaighn, M. E., Malan-Shibley. L., Lechner, J. F., Rhim. J. S.. andHarris, C. C. Human bronchial epithelial cells neoplastically transformed by v-Ki-ros:altered response to inducers of terminal squamous differentiation. Oncogene Res., 3:401^408, 1988.Pfeifer, A. M. A., Mark, G. E., Malan-Shibley. L.. Graziano, S.. Amslad, P., andHarris, C. C. Cooperation of c-ra/-l and c-mvr protooncogenes in the neoplastictransformation of simian virus 40 large tumor antigen-immortalized human bronchialepithelial cells. Proc. Nati. Acad. Sci. USA. 86: 10075-10079, 1989.Gerwin, B. I., Spillare. E., Forrester, K., Lehman, T. A., Kispert, J., Welsh, J. A.,Pfeifer. A. M. A., Lechner, J. F., Baker. S. J., Vogelstein, B., and Harris. C. C. Mutantp53 can induce tumorigenic conversion of human bronchial epithelial cells and reducetheir responsiveness to a negative growth factor, transforming growth factor ÃŸ|.Proc.Nati. Acad. Sci. USA, 89: 2759-2763, 1992.Broad, P. M., Symes. A. J., Thakker, R. V., and Craig, R. K. Structure and methylationof the human calcitonin/a-CGRP gene. Nucleic Acids Res., 17: 6999-7011. 1989.Feinberg, A. P., and Vogelstein, B. A. A technique for radiolabeling DNA restrictionendonuclease fragments to high specific activity. Anal. Biochem., 132: 6-13, 1983.Seiden, R. F. Analysis of DNA sequences by blotting and hybridization. In: F. A.Ausubel, R. Brent, R. E. Kingston. D. D. Moore, J. G. Seidman, J. A. Smith, and K.Strahl (eds.). Current Protocols in Molecular Biology, pp. 2.9.1-2.9.10. New York:Greene Publishing and Wiley-Interscience, 1989.Shmookler Reis. R. J., and Goldstein. S. Interclonal variation in methylation patternsfor expressed and non-expressed genes. Nucleic Acids Res., 14: 4293^304, 1982.

1688



ALTERED DNA METHYLATION IN HUMAN BRONCHIAL EPITHELIAL CELLS

23. Nakamura, V. Leppert. M., O'Connell. P.. Wolff. R.. Holm. T.. Culver, M.. Martin, C.,

Fujimoto. E., Hoff, M.. Kumlin. E., and White, R. Variable number of tandem repeat(VNTR) markers for human gene mapping. Science {Washington DC). 235: 1616-

1622. 1987.24. Ledbetter. S. A.. Wallace. M. R., Collins, F. S., and Ledbetter. D. H. Human chro

mosome 17 /VÂ»illinking clones and their use in long-range restriction mapping of theMiller-Dieker chromosome region (MDCR) in I7pl3.3. Genomics. 7: 264-269. 1990.

25. Lindsay. S., and Bird. A. P. Use of restriction enzymes to detect potential genesequences in mammalian DNA. Nature (Lond.l, 327: 336-338, 1987.

26. Pfeifer. A. M. A.. Jones. R. T.. Bowden. P. E., Mann, D., Spillare. E., Klein-Szanto,

A. J. P.. Trump, B. F.. and Harris. C. C. Human bronchial epithelial cells transformedby the c-ra/-l and c-mvr protooncogenes induce multidifferentiated carcinomas innude mice: a model for lung carcinogenesis. Cancer Res., 51: 3793-3801. 1991.

27. Toth, M.. Lichtenberg. U., and Doerfler. W. Genomic sequencing reveals a 5-meth-ylcytosine-free domain in active promoters and the spreading of preimposed methy-lation patterns. Proc. Nati. Acad. Sci. USA, 86: 3728-3732, 1989.

28. Ledbetter, D. H.. Ledbetter, S. A.. vanTuinen. P.. Summers, K. M.. Robinson, T. J..Nakamura. Y. Wolff, R., White, R., Barker. D. F., Wallace. M. R., Collins, F. S., andDobyns, W. B. Molecular dissection of a contiguous gene syndrome: frequent sub-microscopic deletions, evolutionarily conserved sequences, and a hypomethylated"island" in the Miller-Dieker chromosome region. Proc. Nati. Acad. Sci. USA. 86:

5136-5140, 1989.

29. Sozzi, G., Miozzo, M., Donghi, R., Piloni. S., Cariani. C. T., Pastorino, U., DellaPorta. G., and Pierotti. M. A. Deletions of 17p and p53 mutations in preneoplasticlesions of the lung. Cancer Res.. 52: 6079-6082. 1992.

30. Yokota. J., Wada, M., Shimosato, Y., Terada, M., and Sugimura. T. Loss of heterozy-gosity on chromosomes 3. 13, and 17 in small-cell carcinoma and on chromosome 3in adenocarcinoma of the lung. Proc. Nati. Acad. Sci. USA. 84: 9252-9256. 1987.

31. Weston, A., Willey. J. C.. Modali. R.. Sugimura, H.. McDowell, E. M., Resau, J.,Light, B.. Haugen, A.. Mann, D. L.. Trump, B. F., and Harris, C. C. Differential DNAsequence deletions from chromosome 3, II, 13. and 17 in squamous-cell carcinoma,large-cell carcinoma, and adenocarcinoma of the human lung. Proc. Nati. Acad. Sci.

USA. 86: 5099-5103. 1989.

32. Fults, D., Tippets, R. H., Thomas, G. A., Nakamura. Y., and White, R. Loss ofheterozygosity for loci on chromosome 17p in human malignant astrocytoma. CancerRes., 49: 6572-6577, 1989.

33. Baker, S. J., Fearon, E. R., Nigro, J. M., Hamilton, S. R.. Preisinger. A. C.. Jes.sup, J.M., vanTuinen. P.. Ledbetter, D. H., Barker, D. F., Nakamura, Y, White, R., andVogelstein. B. Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science (Washington DC). 244: 217-221, 1989.

34. Tsai, Y. C.. Nichols, P. W., Hiti, A. L.. Williams. Z., Skinner, D. G., and Jones, P. A.Allelic losses of chromosomes 9, 11, and 17 in human bladder cancer. Cancer Res.,50: 44-47, 1990.

35. Sato. T., Tanigami, A., Yamakawa, K., Akiyama, F., Kasumi. F., Sakamoto, G., andNakamura, Y. Allelotype of breast cancer: cumulative alÃelelosses promote tumorprogression in primary breast cancer. Cancer Res., 50: 7184â€”7189, 1990.

36. Naylor, S. L., Johnson, B. E., Minna, J. D., and Sakaguchi, A. Y. Loss of heterozygosity of chromosome 3p markers in small-cell lung cancer. Nature {Lond.K 329:451-454, 1987.

37. Rabbitts, P., Douglas. J.. Daly, M., Sundaresan, V.. Fox, B., Haselton. P., Wells, F.,Albertson, D., Waters, J., and Bergh, J. Frequency and extent of alleile loss in the shortarm of chromosome 3 in nonsmall-cell lung cancer. Genes Chromosomes Cancer. I:95-105, 1989.

38. Yokoyama, S.. Yamakawa. K., Tsuchiya, E., Murata, M., Sakiyama, S.. and Nakamura, Y. Deletion mapping on the short arm of chromosome 3 in squamous cellcarcinoma and adenocarcinoma of the lung. Cancer Res., 52: 873-877, 1992.

39. Hibi, K.. Takahashi, T.. Yamakawa, K., Ueda, R., Sekido, Y, Ariyoshi, Y, Suyama.M., Takagi, H.. and Nakamura. Y. Three distinct regions involved in 3p deletion inhuman lung cancer. Oncogene, 7: 445â€”449,1992.

40. Sakai, T., Toguchida, J., Ohtani. N., Yandell. D. W., Rapaport. J. M., and Dryja, T. P.Ailele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am. J.Hum. Genet., 48: 880-888, 1991.

41. Keshet, I., Yisraeli, J., and Cedar, H. Effect of regional DNA methylation on geneexpression. Proc. Nati. Acad. Sci. USA, 82: 2560-2564, 1985.

1689



1993;53:1684-1689. Cancer Res Paula M. Vertino, Elisa A. Spillare, Curtis C. Harris, et al. Epithelial CellsOncogene-induced Transformation of Human Bronchial Altered Chromosomal Methylation Patterns Accompany

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