5q11, 8p11, and 10q22 Are Recurrent ChromosomalBreakpoints in Prostate Cancer Cell Lines

Yi Pan,1,2 Weng-Onn Lui,1 Nina Nupponen,3 Catharina Larsson,1* Jorma Isola,3 Tapio Visakorpi,3

Ulf S.R. Bergerheim,2 and Soili Kytola1,3

1Department of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden2Department of Urology, Karolinska Hospital, Stockholm, Sweden3Laboratory of Cancer Genetics, Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere,Finland

Prostate cancer cell lines have been widely used as model systems characterizing pathogenetic, functional, and therapeuticaspects of prostate cancer development. However, their chromosomal compositions are poorly characterized. In this study,five prostate cancer cell lines—TSU-Pr1, JCA-1, NCI-H660, ALVA-31, and PPC-1—were investigated by G-banding, com-parative genomic hybridization (CGH), and spectral karyotyping. The results were combined with our previous findings in theprostate cancer cell lines PC-3, DU145, and LNCaP. By comparative genomic hybridization (CGH), the most frequent losseswere observed at 13q, 8p, 9p, and 4q, whereas gains were most commonly seen at 8q, 10q, and 18p. The composite karyotypeswere characterized by multiple numerical and structural chromosomal aberrations. Recurrent breakpoints at 5q11, 8p11, and10q22 were observed to participate in deletion and translocation events in five of the cell lines, suggesting the importance oftumor suppressor and/or oncogenes in these regions. ALVA-31 and PPC-1 shared nine identical derivative chromosomes, twoof which have also been detected in PC-3. In addition, the identification of the same homozygous deletion at D10S541 and ofan identical TP53 gene mutation in all three cell lines suggests a common origin of these cell lines. © 2001 Wiley-Liss, Inc.

INTRODUCTION

Prostate cancer is the most common cancer inthe Western male population, and the incidence isconstantly increasing. Extensive molecular studieshave shown many genes to be involved in prostatetumorigenesis; however, the precise participationof these genes has not yet been demonstrated.

The importance of chromosomes 7, 8, 10, 13, 16,17, and the X chromosome in prostate tumorigen-esis has been well documented in studies of nu-merical chromosomal alterations by comparativegenomic hybridization (CGH) and loss of heterozy-gosity (LOH) analyses (Carter et al., 1990; Kunimiet al., 1991; Joos et al., 1995; Visakorpi et al., 1995;Cher et al., 1996). The most common alterationsare loss of 8p and gain of 8q, which have beendetected in more than 80% and 90% of the cases,respectively (Visakorpi et al., 1995; Cher et al.,1996; Vocke et al., 1996). Gain of 8q is often asso-ciated with loss of 8p, especially in hormone-refrac-tory carcinomas and in distant metastases (Visa-korpi et al., 1995; Cher et al., 1996). Otherfrequently reported numerical alterations includelosses of 10q, 13q, 16q, and 17 as well as gains of 7,17q, and Xq.

Because of difficulties in cell culturing, only fewcytogenetic studies have been performed on pros-tate carcinomas (Brothman et al., 1990; Lundgrenet al., 1992; Sandberg, 1992; Brothman, 1997). Most

cases have shown normal male karyotypes (46,XY).The most common numerical alterations that havebeen detected are 2Y and 17, whereas structuralchanges have been detected mainly on chromo-somes 1, 7, 8, and 10. Many chromosomal aberra-tions in prostate carcinomas remain undetected byconventional G-banding because of the poor qual-ity of tumor metaphase cells and the complexkaryotypes with many marker chromosomes. Newmolecular cytogenetic techniques, such as spectralkaryotyping (SKY), have been developed to solvesome problems of identification of aberrant chro-mosomes (Schrock et al., 1996). SKY is based onthe simultaneous hybridization of 24 differentlylabeled chromosome-specific painting probes onmetaphase cells, allowing the simultaneous visual-ization of all human chromosomes in different col-ors. SKY has been used successfully in cancer re-search as a tool to identify the chromosomalcomposition of numerical aberrations as well asstructural alterations such as subtle rearrange-

Grant sponsor: Swedish Cancer Foundation; Grant sponsor: Swed-ish Torsten and Ragnar Soderberg Foundation.

*Correspondence to: Catharina Larsson, Department of Molecu-lar Medicine, Endocrine Tumor Unit, Karolinska Hospital, CMML8:01, S-171 76 Stockholm, Sweden.E-mail: catharina.larsson@cmm.ki.se

Received 19 May 2000; Accepted 28 July 2000

GENES, CHROMOSOMES & CANCER 30:187–195 (2001)

© 2001 Wiley-Liss, Inc.

ments, complex translocations, marker chromo-somes, double minutes, and ring chromosomes(Ghadimi et al., 1999; Macville et al., 1999; Padilla-Nash et al., 1999; Pan et al., 1999; Adeyinka et al.,2000; Gray et al., 2000; Kytola et al., 2000). The useof SKY combined with conventional banding andother molecular cytogenetic techniques, such asCGH, makes it possible to identify and resolvealmost all chromosomal aberrations, with the ex-ception of very small changes that are beyond theresolution of these methods.

In this study, we have characterized the karyo-types of five frequently used prostate cancer celllines by G-banding, CGH, SKY, and fluorescencein situ hybridization (FISH) with whole-chromo-some painting probes.

MATERIALS AND METHODS

Cell Lines

Four established human prostate cancer celllines—TSU-Pr1, JCA-1, ALVA-31, and PPC-1—were kindly provided by Dr. Marion J.G. Busse-makers, University Hospital, Nijmegen, the Neth-erlands. TSU-Pr1 was established from a prostaticadenocarcinoma metastatic to lymph nodes (Iizumiet al., 1987), JCA-1 was derived from a poorly tomoderately differentiated prostatic adenocarci-noma (Muraki et al., 1990), ALVA-31 from a well-differentiated adenocarcinoma of the prostate(Loop et al., 1993), and PPC-1 from a poorly dif-ferentiated adenocarcinoma of the prostate (Broth-man et al., 1989). The cells were cultured in RPMI1640 (GIBCO, New York, NY) with 10% fetal bo-vine serum (FBS). The NCI-H660 (CRL-5813)prostate cancer cell line was obtained from theAmerican Type Culture Collection (ATCC; Rock-ville, MD) and cultured in RPMI growth mediumwith the additives recommended by the ATCC,supplemented with 5% FBS. NCI-H660 was de-rived from a small-cell prostate tumor metastatic tolymph nodes.

Harvesting, preparation of metaphase slides, andstaining for G-banding were done according to stan-dard protocols (Mandahl, 1992). The clonality criteriaand the description of karyotypes followed the rec-ommendations of the ISCN (ISCN, 1995). GenomicDNA was extracted for CGH using proteinase-K di-gestion and phenol-chloroform-isoamylalcohol ex-traction.

Comparative Genomic Hybridization

CGH was carried out and the results were ana-lyzed as described (Kallioniemi et al., 1992). Eight

to 12 ratio profiles were averaged for each chromo-some (except the Y chromosome), and green-to-red(g/r) fluorescence ratios of ,0.85 were scored aslosses, those exceeding 1.15 as gains, and regionalaberrations with a g/r ratio .2 as high-level ampli-fications.

Spectral Karyotyping (SKY)

A probe cocktail containing 24 differentially la-beled chromosome-specific painting probes andCot-1 blocking DNA (SKY kit, ASI Applied Spec-tral Imaging, Migdal Ha’Emek, Israel) was dena-tured and hybridized to denatured tumor meta-phase chromosomes according to the protocolrecommended by ASI. After hybridization andwashing, the chromosomes were counterstainedwith DAPI. Image acquisitions were performedusing a SD200 Spectracube system (ASI) mountedon a Zeiss Axioskop microscope with a custom-designed optical filter (SKY-1, Chroma Technol-ogy, Brattleboro, VT). The conversion of emissionspectra to the display colors was achieved by as-signing blue, green, and red colors to specific sec-tions of the emission spectrum. For each cell line,at least 14 metaphase cells were analyzed. SKYresults were confirmed with conventional two-colorFISH.

Deletion Analysis at the D8S1802 andD10S541 Loci

The microsatellite markers D8S1802 at 8q23 andD10S541 at 10q23.3 were used for deletion analysisin the cell lines LNCaP, PC-3, ALVA-31, andPPC-1. Primer sequences for D8S1802 (59-ACATCTGCCTCTCAATTTT-39 and 59-TG-CAGCCCTCAAAGAC-39) and D10S541 (59-AAG-CAAGTGAAGTCTTAGAACCACC-39 and 59-CCACAAGTAACAGAAAGCCTGTCTC-39), andpolymerase chain reaction (PCR) conditions wereobtained from the Whitehead Institute database.The PCR products were separated on a 1.2% aga-rose gel.

Sequencing of Exon 5 of the TP53 Gene

Exon 5 of the TP53 gene was amplified by PCRwith the primers p53 5A (59-TTCAACTCT-GTCTCCTTC-39), and p53 5B (59-GCAAT-CAGTGAGGAATCA-39) and sequenced using anABI PRISM Dye Terminator Cycle SequencingReady Reaction kit (Perkin-Elmer, Foster City,CA) and an ABI310 sequencer (Perkin-Elmer).

188 PAN ET AL.

RESULTS

Comparative Genomic Hybridization

The copy number imbalances identified in thefive prostate cancer cell lines used in this study andin PC-3, DU145, and LNCaP from the previousstudy (Nupponen et al., 1998) are illustrated inFigure 1 and detailed for each case in Table 1. Themean number of CGH alterations was 21.1 6 10.2,mean 6 SD (range 4–35), per cell line. Losses werealmost as common as gains (11.1 6 4.9 vs. 10.0 65.9). The most commonly seen losses were de-tected at 13q21 (88%), 8cen-p12 (75%), 9p21-pter(75%), and 4q22-qter (63%), whereas gains morefrequently involved 8q23–24 (75%), 10q22 (63%),and 18p (63%). High-level amplifications wereseen at 20q and 8q23-qter in the TSU-Pr1 andALVA-31 cell lines, respectively, as well as at10p12–q23 and 14q12–24 in PC-3.

Karyotyping

All the cell lines were found to be near-triploid,with chromosome numbers ranging from 61-84(modal numbers 66–79, Table 2). The karyotypes

based on G-banding, CGH results, and SKY anal-ysis are shown in Table 2. Several complex trans-locations including chromosomal breakpoints up toseven were detected. Nine isochromosomes wereidentified, including i(1)(p10) in TSU-Pr1,i(5)(p10), i(13)(q10), and i(17)(p10) in JCA-1,i(18)(p10), and i(22)(p10) in ALVA-31, andi(5)(p10), i(18)(p10), and i(18)(q10) in PPC-1. TheY chromosome, double minutes, or ring chromo-somes were not detected in any of the cell lines.

Comparison of Karyotypes and Breakpoints

The karyotypes and breakpoints of the five celllines analyzed in this study were compared withthe previously published results for the prostatecancer cell lines DU145, LNCaP, and PC-3 (Pan etal., 1999; Beheshti et al., 2000). The derivativechromosomes were mainly the same in these celllines, especially in DU145 and LNCaP, in bothstudies, but the breakpoints were slightly different.The cell lines ALVA-31 and PPC-1 shared nineidentical derivative chromosomes, two of whichhave also been detected in PC-3 (Table 3). Exceptin the common derivative chromosomes in ALVA-

Figure 1. Summary of DNA copy number alterations detected by CGH in eight prostate cancer celllines. Each line represents one alteration detected in one cell line, with losses illustrated to the left and gainsto the right of the ideograms. High-level amplifications are shown by bold lines.

189RECURRENT BREAKPOINTS IN PROSTATE CANCER

31, PPC-1, and PC-3, the only rearrangements seenrecurrently in at least two cell lines were theabnormal chromosomes der(8)t(7;8)(q32;p11.2),del(8)(p11.1), i(5)(p10), del(9)(p22), and der(18)-t(15;18)(q22;p11.2). Eight recurrent breakpointswere found in at least four of the cell lines. Thebands 5q11, 8p11, and 10q22 were involved inrearrangements in five cell lines each (Fig. 2),whereas 5p11, 10p11, 12q24, 15q15, and 17q25were rearranged in four cell lines each. The chro-mosomal arms 8p and 10q were the most fre-quently altered in the CGH analyses.

Deletion Analysis of D10S541 and Sequencing ofthe TP53 Gene

Homozygous deletion of the marker D10S541was detected in PC-3, PPC-1, and ALVA-31,whereas in LNCaP one amplified band was de-tected. Marker D8S1802 was used as a control ofPCR amplification, which, in all cases, resulted in aPCR product of the expected size. A single basedeletion at codon 169 in exon 5 of the TP53 genewas found in PC-3, PPC-1, and ALVA-31, whereasin LNCaP the sequence was normal.

DISCUSSION

The prostate cancer cell lines TSU-Pr1, JCA-1,NCI-H660, ALVA-31, and PPC-1 have been

widely used in pathogenetic and functional pros-tate studies, but the cytogenetic composition ofthese cell lines has been poorly characterized. Par-tial karyotypes of these cell lines (Iizumi et al.,1987; Brothman et al., 1989; Johnson et al., 1989;Muraki et al., 1990; Brothman and Patel, 1992;Loop et al., 1993) have been reported previously,with many unidentified and poorly characterizedchromosomes. In this study, the composition of allthe chromosomes in five prostate cancer cell lineswas identified, clarifying many previously unchar-acterized chromosomes. The chromosome num-bers matched well with previous reports based onG-banding, suggesting that the cell lines have re-mained stable after repeated subculturing. How-ever, for TSU-Pr1, a slight discrepancy was de-tected between the karyotype originally reportedand the karyotype described in this study. Of theoriginal karyotype of 80,XXY,t(6;13)(q15;q32),i(1p),i(1q), inv(3)(p25;q11),8p2,10q2,17p1,20q1,10mar (Iizumi et al., 1987), only i(1p), 8p2, 17p1,and 20q1 were found in this study. The reason forthe difference might be the different interpretationof the chromosomes, with the poor quality of theG-bands in the original study or the complexity ofthe rearranged chromosomes. Double minuteswere not detected in PPC-1 in this study, whereas

TABLE 1. Chromosomal Imbalances in the Eight Prostate Cancer Cell Lines Detectedby Comparative Genomic Hybridization (CGH)

Cell line Losses Gainsb

NCI-H660 2q21–32, 4q21-qter, 5q14–23, 7q32-qter, 8p,13cen-q21, 15cen-q21, 16q, 22q

6, 7pter-q21, 8q, 10pter-q22

TSU-Pr1 Xp11.3-pter, 3cen-p14, 7q21–31, 8p,9p21-pter

Xq, 1p, 5q31-qter, 11p14-pter, 11cen-q14, 16p, 17,19pter-q13.1, 20 (20q)

JCA-1 X, 1q24–31, 5cen-q15, 6p, 7p14–q31, 8p,9p21-pter, 11cen-p14, 13cen-q31, 14cen-q12, 18q22-qter

1q41-qter, 5p14-pter, 5q23-qter, 7q32-qter, 8q,10q22-23, 16q, 17p, 18p, 20

ALVA-31 1p21–22, 1q25–31, 2q22, 3p13–14, 4q22-qter,5cen-q22, 6cen-q22, 7p14–15, 8p12–q21,9p21-pter, 10p, 10q23–24, 13q, 18q, 21q,22q

Xp11.3-q13, Xq25-qter, 1cen-q23, 2q34-qter, 3q26-qter, 5p14-pter, 5q32-qter, 7q32-qter, 8q23-qter, 9q22-qter, 10q21–22, 12p12–q13, 12q22-qter, 14q22-qter, 16, 17q22-qter, 18p, 19p, 20q

PPC-1 3cen-p13, 4cen-p15.1, 4q21-qter, 5cen-q13,8p, 9p13-pter, 10p12-pter, 11q13-qter,13q, 15q15-qter, 18q12, 20p

Xq, 1p33-pter, 1cen-q23, 1q32, 2p21-pter, 5p, 8q13-qter, 9q31-qter, 10p11.2-q22, 14q, 17q21-qter,18p, 18q21-qter, 19

PC-3a 4p, 4q21-qter, 5cen-q15, 5q23-q31, 6q, 8pter-q12, 9pter-q34, 10p13-pter, 10q24-qter,12p12-pter, 13q21, 15q21-qter, 16, 17pter-q12, 18q12–21, 19cen-q13, 22q

Xp22.2-qter, 1q21–25, 1q31-qter, 7pter-q35, 8q13-qter, 10p12–q23, 11q13-qter, 12q21-qter,14q12–24, 17q21-qter, 18p, 20pter-q13.2

DU145a Xp21-qter, 1p, 2q, 3pter-q22, 4cen-q31,4q31-qter, 6q, 9p21-pter, 10p12, 11p,12p12-pter, 13q, 16p, 18q21-qter, 20p

1q, 2p13-pter, 3q24-qter, 4p15-pter, 5p, 8cen-q24,9p13–q21, 11q14-qter, 12q21–23, 14q, 18pter-q12, 21q

LNCaPa 1p34-pter, 2p13–22, 6cen-q16, 13q14-qter —

aData from a previously published study (Nupponen et al., 1998).bThe high-level amplifications are listed in bold.

190 PAN ET AL.

TA

BLE

2.K

aryo

type

sof

the

Five

Pros

tate

Can

cer

Cel

lLin

esA

naly

zed

inT

his

Stud

y

Cel

llin

eK

aryo

type

base

don

G-b

andi

ng,C

GH

,SK

Y,a

ndFI

SHM

odel

no.

TSU

-Pr1

64;

78^3

n&,X

,der

(X)t

(X;1

0)(p

11.2

;q23

),2Y

,i(1)

(p10

),der

(1;5

)(q1

0;q1

0),1

der(

1)t(

1;7)

(q25

;q22

),der

(2)t

(2;3

)(q3

3;p2

1),

der(

2)t(

2;20

)(p1

1.2;

p11.

2)t(

2;15

)(q3

3;q2

4),1

der(

2)t(

2;15

)(q3

3;q2

4),d

er(3

)t(3

;19)

(p11

;p11

.2),d

er(5

;20)

(p10

;q10

),del

(7)(

q11.

1),

der(

8)t(

7;8)

(q32

;p11

.2),d

el(9

)(p2

2),d

er(9

)t(9

;11)

(q34

;q13

)?du

p(11

)(q2

3q25

),der

(10)

t(10

;20)

(p11

.2;p

11.2

),del

(11)

(q23

),1

del(1

1)(q

13),1

12,d

el(1

5)(q

21),d

er(1

5)t(

2;15

)(q3

3;q1

5),d

er(1

5)t(

5;15

)(q3

1;p1

1.1)

,1de

r(16

;17)

(p10

;p10

),1de

r(17

)t(3

;17)

(q11

.2;q

25),

1de

r(17

)t(2

;17)

(q11

.2;q

11.1

)t(2

;3)(

q33;

p21)

,1de

r(17

)t(1

5;17

)(q2

4;q1

1.1)

,der

(18)

t(15

;18)

(q22

;p11

.2)x

2,1

18,1

20x2

[cp1

4]

76

JCA

-163

;69

^3n&

,X,2

X,2

Y,d

el(1

)(q2

3q32

),der

(1)t

(1;1

7)(p

13;q

11.2

),1de

r(1;

17)(

p10;

p10)

,der

(2;1

0)(q

10;p

10),1

del(2

)(q1

1.1)

,del

(4)(

q25)

,i(5

)(p1

0),d

er(5

)t(5

;11)

(p11

;q13

)x2,

1de

r(5;

10)(

p10;

q10)

,1id

er(5

)(q1

0)de

l(5)(

q11.

2q22

),1de

r(5)

t(5;

10)(

p15.

3;q2

2)de

l(5)(

q11.

1),

der(

6;20

)(q1

0;q1

0),2

7,de

r(8)

t(7;

8)(q

32;p

11.2

)x2,

18,

del(9

)(p1

3),d

el(1

0)(q

11.1

),der

(10)

t(7;

10)(

?;p13

)t(2

;7)(

?;?),d

er(1

1;20

)(p1

0;p1

0),

211

,der

(13)

t(10

;13)

(q11

.2;q

12),i

(13)

(q10

),213

,214

,del

(15)

(q22

)x2,

der(

16)t

(11;

16)(

?p15

;p13

.1),1

der(

16)t

(10;

16)(

q11.

1;p1

1.1)

t(1;

10)(

q32;

q24)

,i(17

)(p1

0),2

17,d

er(1

8)t(

13;1

8)(q

32;q

21),d

er(1

8)t(

15;1

8)(q

22;p

11.2

),1de

r(20

;21)

(q10

;q10

),der

(21)

t(17

;21)

(q11

.2;q

11.2

),221

,der

(22)

t(18

;22)

(?;p

11.2

)t(1

5;?1

8)(q

22;?)

,der

(22)

t(14

;22)

(q13

;p11

.2)

[cp1

7]

67

ALV

A-3

166

;74

^3n&

,XX

,1de

l(X)(

q21)

,2Y

,der

(1)t

(1;1

0)(q

25;q

23),d

er(1

)t(1

;20)

(p11

;?q12

)t(1

;10)

(q21

;?),1

der(

1,8,

10,1

2,18

)(18

qter

2.

q11.

2<12

?<8?

<10

?<12

?<8?

<10

?<1p

112

.qt

er),1

der(

1)(8

qter

2.

q13<

12?<

1?<

12?<

1p36

.32

.q2

2<10

?<3q

122

.qt

er),

der(

2)t(

2;8)

(p23

;q24

),der

(2)t

(15;

17)(

q11.

2;q2

1)t(

2;15

)(p2

5;q1

5),d

er(2

)t(2

;7)(

p25;

p21)

,der

(3)t

(3;1

0)(q

11.1

;?p11

.2)x

2,2

3,de

r(4)

t(4;

9)(q

35;q

22)t

(3;9

)(?q

25;q

34),1

der(

4)t(

4;5)

(p11

;p12

)t(4

;14)

(q21

;q23

),1de

r(4)

t(4;

20)(

p11;

q11.

2)de

l(4)(

q13)

,der

(5)t

(5;1

5)(q

11.2

;q13

),25,

1de

r(7)

t(2;

15)(

p25;

q11.

2q15

)t(2

;7)(

p11.

2;p1

1.1)

,28,

der(

9)t(

9;19

)(p2

4;?)

t(5;

19)(

q11.

2;?)

,1de

r(9)

(3qt

er2

.?q

25<

17q2

12.

q25<

15q1

1.22

.q1

5<9p

112

.qt

er),d

el(1

0)(?

),der

(1,3

,10)

(3pt

er2

.p1

1<10

?<1q

?2.

qter

<10

?<1q

?2.

qter

<10

?<

3q11

.22

.qt

er),d

er(1

0)(8

qter

2.

q12<

12?<

10?<

1?<

3?),d

er(1

2)t(

12;2

2)(q

11;q

11.1

),der

(12)

t(10

;12)

(?;q

22)t

(5;1

0)(q

22;?)

,de

r(12

)t(1

2;20

)(q2

4;?)

,1de

r(12

)t(8

;12)

(q22

;p12

)t(8

;12)

(q22

;q15

),1de

r(12

)i(12

)(q1

0)de

l(12)

(q22

),der

(13)

ins(

13;2

1)(q

32;?)

t(13

;17)

(p11

;p11

),213

,114

,der

(15)

t(5;

15)(

q13;

p13)

,215

,215

,1de

r(16

)t(1

6;19

)(q2

2;?)

,217

,der

(18)

t(1;

18)(

?;q12

)t(1

;10)

(?;?q

22),

i(18)

(p10

),1de

r(18

)t(2

;18)

(q31

;q11

.2),d

er(1

9)(1

6?<

19?<

10?<

5?),d

er(2

0)t(

15;2

0)(q

11.2

;p13

),221

,222

,?i(2

2)(p

10)

[cp1

7]

66

PPC

-161

;84

^3n&

,XX

,1de

r(X

)t(X

;19)

(p11

.1;q

11),2

Y,d

er(1

)t(1

;4)(

p36.

3;q2

5)t(

1;10

)(q2

1;q2

2),d

er(1

)(8q

ter2

.q1

3<12

?<1p

36.3

2.

q25<

10q2

42.

qter

),1de

r(1;

2)(q

10;q

10),1

ider

(1)(

q10)

t(1;

10)(

q21;

q22)

,del

(2)(

q23)

,der

(2)t

(2;8

)(p2

5;q1

3),d

er(2

)t(1

5;17

)(q

11.2

;q21

)t(2

;15)

(p25

;q15

),1de

r(2)

t(2;

7)(p

25;p

21),d

er(3

)t(3

;10)

(q11

.1;?p

11.2

),23,

23,

der(

4)(5

pter

2.

p11<

4p11

2.

q21<

10?

<13

q112

.qt

er),1

der(

4)t(

4;20

)(p1

1;q1

1.2)

del(4

)(q1

3)x2

,1id

er(4

)(q1

0)t(

4;10

)(q2

1;q2

2),i(

5)(p

10),d

er(5

)t(5

;15)

(q11

.2;q

13),

1de

r(6)

t(6;

7)(p

22;?)

t(7;

8)(?

;q22

),del

(8)(

p11.

1),d

el(9

)(p2

2),1

der(

9)t(

9;18

)(p1

1;p1

1.2)

,1de

l(9)(

?),d

er(1

,3,1

0)(3

pter

2.

p11<

10?<

1q?2

.qt

er<

10?<

1q?2

.qt

er<

10?<

3q11

.22

.qt

er)x

2,de

r(10

)(16

?<10

?<9?

<3q

122

.qt

er),1

der(

11)t

(11;

20)(

q12;

p11.

2),1

der(

?11)

(16p

ter2

.p1

1<11

?<15

q112

.q1

5<17

q212

.q2

5<10

?),d

er(1

2)t(

12;2

0)(q

24;?)

x2,d

er(1

2)t(

8;12

)(q1

3;q2

4),1

der(

12)t

(7;1

2)(?

;?),2

13,1

14,1

14,1

14,

der(

15)t

(5;1

5)(q

13;p

13),2

15,2

15,d

er(1

7)t(

1;17

)(p2

2;q2

5),i(

18)(

p10)

,1i(1

8)(q

10),d

er(1

9;22

)(p1

0;q1

0),1

der(

19)t

(18;

19)

(p11

;?)t(

5;19

)(q1

3;?)

x2,d

er(2

0)t(

13;2

0)(?

;p11

.1)t

(8;1

3)(?

;?),2

20,1

der(

21)t

(3;2

1)(?

;p11

.1),2

22[c

p15]

79

NC

I-H66

066

;70

^3n&

,X,d

er(X

)t(X

;8)(

q22;

q24)

,2Y

,del

(1)(

p13p

31),d

er(2

)t(2

;3)(

p25;

q23)

,der

(2)t

(2;3

)(q3

3;q2

5),2

2,de

l(3)(

q25)

x2,d

er(3

)t(3

;7)(

q25;

p15)

,1

der(

3)t(

3;5)

(q25

;q31

)t(2

;5)(

q33;

q35)

,del

(4)(

q23)

,der

(4)t

(4;1

0)(q

11;q

11.2

)t(1

0;15

)(q2

2;q2

2),d

el(5

)(q1

5),d

er(5

)del

(5)(

p12)

del(5

)(q3

1),

der(

5)t(

4;5)

(q22

;p11

)del

(5)(

q14q

23),1

der(

5)t(

3;5)

(q25

;q23

),der

(6)t

(6;1

2)(p

25;q

24)x

2,1

6,de

r(7)

t(7;

20)(

q21;

p11.

2),d

er(7

)t(7

;16)

(p22

;q11

.2)t

(7;2

0)(q

21;p

11.2

),der

(7)d

el(7

)(p1

4)t(

7;18

)(q1

1.1;

q11.

1),1

der(

7)(?

10pt

er2

.p?

<7p

222

.q2

1<18

q11.

22.

q21<

1p22

2.

pter

),de

l(8)(

p11.

1),d

er(1

0)t(

10;1

2)(q

22;q

14)x

2,1

der(

10)t

(1;1

0)(q

12;p

12),d

er(1

2)t(

X;8

)(q2

2;?)

t(8;

12)(

?;q22

),212

,der

(13;

14)(

q10;

q10)

x2,2

13,2

14,

215

,215

,der

(16)

t(7;

16)(

?;q11

.1),2

16,d

er(1

7)t(

17;1

8)(q

11.1

;q21

),1de

r(17

)t(1

7;?1

)(q2

5;?)

,del

(18)

(q11

.2)x

2,de

r(19

)t(8

;19)

(q11

.1;q

13.2

)x2,

119

,der

(20)

t(13

;20)

(q22

;p13

)t(8

;20)

(q11

.1;q

11.1

),der

(20)

(13q

ter2

.q2

2<20

p132

.11

.1<

19?<

8q11

.12

.qt

er),d

er(2

1)t(

7;21

)(q2

2;p1

1.1)

x2,

222

[cp1

4]

68

CG

H,c

ompa

rativ

ege

nom

ichy

brid

izat

ion;

SKY

,spe

ctra

lkar

yoty

ping

;FIS

H,fl

uore

scen

cein

situ

hybr

idiz

atio

n.

191RECURRENT BREAKPOINTS IN PROSTATE CANCER

they were reported to be present in the originalkaryotype (Brothman et al., 1989). However, themarker chromosomes characterized previously inPPC-1 (Brothman and Patel, 1992) were recogniz-able also in our study.

It is intriguing that all cell lines are near-triploid;however, the reason behind this remains unknown.One explanation could be that this is simply due toculturing artifacts as previously suggested (Ohnukiet al., 1980). On the other hand, it could also berelevant for the biological behavior of the tumor, sothat triploid subclones present in the primary tu-mors would have a selective advantage in the es-tablishment of cell lines. A parallel could be drawnfrom some of our previous studies of establishedcell lines of solid tumors in which near-tetraploidkaryotypes were frequently identified (Pan et al.,1999; Adeyinka et al., 2000; Kytola et al., 2000). Insome of these cases, as well as in some associatedprimary tumors, two clones were observed includ-ing a near-diploid and a near-tetraploid. In thetetraploid clones, several of the markers werepresent in duplicate, suggesting that structural andnumerical aberrations of individual chromosomesoccurred as earlier events, which were then fol-lowed by duplications of the entire genome.Hence, there has been a growth advantage for sub-clones with higher chromosome content in the es-tablishment of cell lines.

Numerous chromosomal abnormalities in allchromosomes were detected, suggesting a high de-gree of genetic instability in prostate cancer celllines. It is well known that cell lines usually aregenetically more complex than the correspondingprimary tumors, having secondary nonspecific

changes that are acquired during cell culture. Tak-ing together the results for all eight prostate cancercell lines characterized previously (Pan et al., 1999;Beheshti et al., 2000) and in this study, eight re-current breakpoints were detected. The break-points at 5q11 and 8p11 were detected in bothdeletion and translocation chromosomes, whereasthe others were seen only as a part of translocationevents. This phenomenon was supported by theCGH results, suggesting the importance of tumorsuppressor genes on 5q and 8p and oncogenes on8q, 10q, 5p, 12q, 14q, and 17q in prostate tumori-genesis. Complete or partial loss of 8p has beenreported repeatedly in prostate carcinomas and inprostate intraepithelial neoplasia (Bergerheim etal., 1991; Cher et al., 1994; Emmert-Buck et al.,1995; Visakorpi et al., 1995). Recently, the deletedregion of 8p has been narrowed down to a1–1.5-Mb region in 8p22 (Van Alewijk et al., 1999;Arbieva et al., 2000), but the tumor suppressorgene(s) in this region involved in prostate tumori-genesis remain unknown. The high frequency ofloss at 8p and the fact that the loss has also beendetected in premalignant lesions (Matsuyama etal., 1994; Jenkins et al., 1998) suggest that the lossat 8p might be an early event in prostate carcino-genesis. By CGH, loss of 8p is often associated withgain of 8q, as seen in JCA-1 and NCI-H660, indi-cating possible isochromosome formation, but noi(8q) was detected by SKY in this study. Loss of 5qand gains of 10q, 5p, 12q, 14q, and 17q have alsobeen detected previously in other studies (Joos etal., 1995; Visakorpi et al., 1995). None of the knowngenes in these regions has been found to be theamplified target genes. The identified recurrent

TABLE 3. Common Structural Alterations in the Prostate Cancer Cell Lines PPC-1, ALVA-31, and PC-3

Derivativechromosome

Cell line

PPC-1 ALVA-31 PC-3a

der(2)t(2;15)t(15;17) der(2)t(2;15)(p25;q15)t(15;17)(q11.2;q21)

der(2)t(2;15)(p25;q15)t(15;17)(q11.2;q21) der(2)t(2;15)(p25;q15)t(15;17)(q11.2;q21)

der(15)t(5;15) der(15)t(5;15)(q13;p13) der(15)t(5;15)(q13;p13) der(15)t(5;15)(q13;p13)der(12)t(8;12) der(12)t(8;12)(q13;q24) der(12)t(8;12)(q13;q24)der(1,3,10) der(1,3,10)(3pter2.p11<10?

<1q?2.qter<10?<1q?2.qter<10?<3q11.22.qter)

der(1,3,10)(3pter2.p11<10?<1q?2.qter<10?<1q?2.qter<

10?<3q11.22.qter)der(2)t(2;7) der(2)t(2;7)(p25;p21) der(2)t(2;7)(p25;p21)der(3)t(3;10) der(3)t(3;10)(q11.1;?p11.2) der(3)t(3;10)(q11.1;?p11.2)der(4)t(4;20) der(4)t(4;20)(p11;q11.2)

del(4)(q13)der(4)t(4;20)(p11;q11.2)

del(4)(q13)der(5)t(5;15) der(5)t(5;15)(q11.2;q13) der(5)t(5;15)(q11.2;q13)der(12)t(12;20) der(12)t(12;20)(q24;?) der(12)t(12;20)(q24;?)i(18) i(18)(p10) i(18)(p10)

aFrom the study by Pan et al. (1999).

192 PAN ET AL.

Figu

re2.

Illus

trat

ion

ofth

ech

rom

osom

alal

tera

tions

part

icip

atin

gin

brea

kpoi

nts

invo

lvin

g5q

11,8

p11,

and

10q2

2in

eigh

tpr

osta

teca

ncer

cell

lines

.The

dele

ted

and

tran

sloc

ated

chro

mos

omes

are

seen

asSK

Ydi

spla

yco

lors

(left

),in

vert

edD

API

-ban

ded

(mid

dle)

,and

clas

sifie

dco

lors

(rig

ht).

The

tran

sloc

atio

npa

rtne

rsar

esh

own

asnu

mbe

rsw

ithin

the

clas

sifie

dco

lors

.The

cyto

gene

ticde

scri

ptio

nfo

rea

chch

rom

osom

eis

give

non

the

righ

t.

breakpoints in the present study suggest the im-portance of genes in these specific breakpoints inprostate tumorigenesis.

The cell lines ALVA-31 and PPC-1 had ninecommon derivative chromosomes, three of themquite complex. Surprisingly, two of the derivativechromosomes were also observed in PC-3. Thispossible cell-line cross-contamination betweenPC-3 and PPC-1 has been reported previously(Chen, 1993). All three cell lines had a homozygousdeletion with the marker D10S541, and the sameTP53 gene mutation, suggesting a common originof these cell lines. The homozygous deletion andthe mutation in TP53 have previously been re-ported in PC-3 (Carroll et al., 1993; Vlietstra et al.,1998). Most likely, the PC-3 cells had contami-nated the PPC-1 and ALVA-31 cultures, due to thefact that the PC-3 was established and submitted tothe ATCC in 1979 (Kaighn et al., 1979), before thePPC-1 and ALVA-31 cell lines had been estab-lished.

To compensate for some drawbacks and weak-nesses of cytogenetic techniques when used alone,we characterized five prostate cell lines by usingG-banding, CGH, and SKY together. The mainfinding was the presence of the recurrent chromo-somal breakpoints in the cell lines, which helpsidentify genes in these regions in future studies.

ACKNOWLEDGMENTS

The authors thank Mrs. Arja Alkula for technicalassistance.

REFERENCES

Adeyinka A, Kytola S, Mertens F, Pandis N, Larsson C. 2000.Spectral karyotyping and chromosome banding studies of primarybreast carcinomas and their lymph node metastases. Int J MolMed 5:235–240.

Arbieva ZH, Banerjee K, Kim SY, Edassery SL, Maniatis VS, Hor-rigan SK, Westbrook CA. 2000. High-resolution physical map andtranscript identification of a prostate cancer deletion interval on8p22. Genome Res 10:244–257.

Beheshti B, Karaskova J, Park PC, Squire JA, Beatty BG. 2000.Identification of a high frequency of chromosomal rearrangementsin the centromeric regions of prostate cancer cell lines by sequen-tial Giemsa banding and spectral karyotyping. Mol Diagn 5:23–32.

Bergerheim USR, Kunimi K, Collins VP, Ekman P. 1991. Deletionmapping of chromosomes 8, 10, and 16 in human prostate carci-noma. Genes Chromosom Cancer 3:215–220.

Brothman AR. 1997. Cytogenetic studies in prostate cancer: are wemaking progress ? Cancer Genet Cytogenet 95:116–121.

Brothman AR, Patel AM. 1992. Characterization of 10 marker chro-mosomes in a prostatic cancer cell line by in situ hybridization.Cytogenet Cell Genet 60:8–11.

Brothman AR, Lesho LJ, Somers KD, Wright GL Jr, Merchant DJ.1989. Phenotypic and cytogenetic characterization of a cell linederived from primary prostatic carcinoma. Int J Cancer 44:898–903.

Brothman AR, Peehl DM, Patel AM, McNeal JE. 1990. Frequencyand pattern of karyotypic abnormalities in human prostate cancer.Cancer Res 50:3795–3803.

Carroll AG, Voeller HJ, Sugards L, Gelmann EP. 1993. p53 onco-

gene mutations in three human prostate cancer cell lines. Prostate23:123–134.

Carter BS, Ewing CM, Ward WS, Treiger BF, Aalders TW,Schalken JA, Epstein JI, Isaacs WB. 1990. Allelic loss of chromo-somes 16q and 10q in human prostate cancer. Proc Natl Acad SciUSA 87:8751–8755.

Chen TR. 1993. Chromosome identity of human prostate cancer celllines, PC-3 and PPC-1. Cytogenet Cell Genet 62:183–184.

Cher ML, MacGrogan D, Bookstein R, Brown JA, Jenkins RB,Jensen RH. 1994. Comparative genomic hybridization, allelic im-balance, and fluorescence in situ hybridization on chromosome 8in prostate cancer. Genes Chromosomes Cancer 11:153–162.

Cher ML, Bova GS, Moore DH, Small EJ, Carroll PR, Pin SS,Epstein JI, Isaacs WB, Jensen RH. 1996. Genetic alterations inuntreated metastases and androgen-independent prostate cancerdetected by comparative genomic hybridization and allelotyping.Cancer Res 56:3091–3102.

Emmert-Buck MR, Vocke CD, Pozzatti RO, Duray PH, JenningsSB, Florence CD, Zhuang Z, Bostwick DG, Liotta LA, LinehanWM. 1995. Allelic loss on chromosome 8p12–21 in microdissectedprostatic intraepithelial neoplasia. Cancer Res 55:2959–2962.

Ghadimi BM, Schrock E, Walker RL, Wangsa D, Jauho A, MeltzerPS, Ried T. 1999. Specific chromosomal aberrations and amplifi-cation of the AIB1 nuclear receptor coactivator gene in pancreaticcarcinomas. Am J Pathol 154:525–536.

Gray SG, Kytola S, Lui WO, Larsson C, Ekstrom T. 2000. Modu-lating IGFBP-3 expression by trichostatin A: potential therapeuticrole in the treatment of hepatocellular carcinoma. Int J Mol Med5:33–41.

Iizumi T, Yazaki T, Kanoh S, Kondo I, Koiso K. 1987. Establish-ment of a new prostatic carcinoma cell line (TSU-Pr1). J Urol137:1304–1306.

ISCN. 1995. An international system for human cytogenetic nomen-clature. Mitelman F, editor. Basel: S Karger.

Jenkins R, Takahashi S, DeLacey K, Bergstralh E, Lieber M. 1998.Prognostic significance of allelic imbalance of chromosome arms7q, 8p, 16q, and 18q in stage T3N0M0 prostate cancer. GenesChromosomes Cancer 21:131–143.

Johnson BE, Whang-Peng J, Naylor SL, Zbar B, Brauch H, Lee E,Simmons A, Russell E, Nam MH, Gazdar AF. 1989. Retention ofchromosome 3 in extrapulmonary small cell cancer shown bymolecular and cytogenetic studies. J Natl Cancer I 81:1223–1228.

Joos S, Bergerheim US, Pan Y, Matsuyama H, Bentz M, du ManoirS, Lichter P. 1995. Mapping of chromosomal gains and losses inprostate cancer by comparative genomic hybridization. GenesChromosomes Cancer 14:267–276.

Kaighn ME, Narayan KS, Ohnuki Y, Lechner JF, Jones LW. 1979.Establishment and characterization of a human prostatic carci-noma cell line (PC-3). Invest Urol 17:16–23.

Kallioniemi A, Kallioniemi O-P, Sudar D, Rutovitz D, Gray JW,Waldman F, Pinkel D. 1992. Comparative genomic hybridizationfor molecular cytogenetic analysis of solid tumors. Science 258:818–821.

Kunimi K, Bergerheim US, Larsson IL, Ekman P, Collins VP. 1991.Allelotyping of human prostatic adenocarcinoma. Genomics 11:530–536.

Kytola S, Rummukainen J, Nordgren A, Karhu R, Farnebo F, IsolaJ, Larsson C. 2000. Chromosomal alterations in 15 breast cancercell lines by comparative genomic hybridization and spectralkaryotyping. Genes Chromosomes Cancer 28:308–317.

Loop SM, Rozanski TA, Ostenson RC. 1993. Human primary pros-tate tumor cell line, ALVA-31: a new model for studying thehormonal regulation of prostate tumor cell growth. Prostate 22:93–108.

Lundgren R, Mandahl N, Heim S, Limon J, Henrikson H, Mitel-man F. 1992. Cytogenetic analysis of 57 primary prostatic adeno-carcinomas. Genes Chromosomes Cancer 4:16–24.

Macville M, Schrock E, Padilla-Nash H, Keck C, Ghadimi BM,Zimonjic D, Popescu N, Ried T. 1999. Comprehensive and de-finitive molecular cytogenetic characterization of HeLa cells byspectral karyotyping. Cancer Res 59:141–150.

Mandahl N. 1992. Methods in solid tumour cytogenetics: humancytogenetics—a practical approach. In: Rooney DE, Cz-epulkowski BH, editors. Malignancy and acquired abnormalities.Oxford: IRL Press.

Matsuyama H, Pan Y, Skoog L, Tribukait B, Naito K, Ekman P,Lichter P, Bergerheim US. 1994. Deletion mapping of chromo-some 8p in prostate cancer by fluorescence in situ hybridization.Oncogene 9:3071–3076.

Muraki J, Addonizio JC, Choudhury MS, Fischer J, Eshghi M,

194 PAN ET AL.

Davidian MM, Shapiro LR, Wilmot PL, Nagamatsu GR, ChiaoJW. 1990. Establishment of new human prostatic cancer cell line(JCA-1). Invest Urol 36:79–84.

Nupponen NN, Hyytinen ER, Kallioniemi AH, Visakorpi T. 1998.Genetic alterations in prostate cancer cell lines detected by com-parative genomic hybridization. Cancer Genet Cytogenet 101:53–57.

Ohnuki Y, Marnell MM, Babcock MS, Lechner JF, Kaighn ME.1980. Chromosomal analysis of human prostatic adenocarcinomacell lines. Cancer Res 40:524–534.

Padilla-Nash HM, Nash WG, Padilla GM, Roberson KM, RobertsonCN, Macville M, Schrock E, Ried T. 1999. Molecular cytogeneticanalysis of the bladder carcinoma cell line BK-10 by spectralkaryotyping. Genes Chromosomes Cancer 25:53–59.

Pan Y, Kytola S, Farnebo F, Wang N, Lui WO, Nupponen N, IsolaJ, Visakorpi T, Bergerheim USR, Larsson C. 1999. Characteriza-tion of chromosomal abnormalities in prostate cancer cell lines byspectral karyotyping. Cytogenet Cell Genet 87:225–232.

Sandberg AA. 1992. Chromosomal abnormalities and related eventsin prostate cancer. Hum Pathol 23:368–380.

Schrock E, du Manoir S, Veldman T, Schoell B, Wienberg J, Fer-

guson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D,Garini Y, Ried T. 1996. Multicolor spectral karyotyping of humanchromosomes. Science 273:494–497.

Van Alewijk DC, Van Der Weiden MM, Eussen BJ, Van DenAndel-Thijssen LD, Ehren-Van Eekelen CC, Konig JJ, VanSteenbrugge GJ, Dinjens WN, Trapman J. 1999. Identification ofa homozygous deletion at 8p12–21 in a human prostate cancerxenograft. Genes Chromosomes Cancer 24:119–126.

Visakorpi T, Kallioniemi AH, Syvanen AC, Hyytinen ER, Karhu R,Tammela T, Isola JJ, Kallioniemi O-P. 1995. Genetic changes inprimary and recurrent prostate cancer by comparative genomichybridization. Cancer Res 55:342–347.

Vlietstra RJ, van Alewijk DCJG, Hermans KGL, van SteenbruggeGJ, Trapman J. 1998. Frequent inactivation of PTEN in prostatecancer cell lines and xenografts. Cancer Res 58:2720–2723.

Vocke CD, Pozzatti RO, Bostwick DG, Florence CD, Jennings SB,Strup SE, Duray PH, Liotta LA, Emmert-Buck MR, LinehanWM. 1996. Analysis of 99 microdissected prostate carcinomasreveals a high frequency of allelic loss on chromosome 8p12–21.Cancer Res 56:2411–2416.

195RECURRENT BREAKPOINTS IN PROSTATE CANCER