Transfer of chromosome 8 into two breast cancer cell lines:total exclusion of three regions indicates location of
putative in vitro growth suppressor genes
Peter Wilson
a
, Andrew Cuthbert
b
, Anna Marsh
a
, Jeremy Arnold
a
, James Flanagan
a
,Christine Mulford
a
, Deborah Trott
b
, Elizabeth Baker
b
, David Purdie
a
,Robert Newbold
b
, Georgia Chenevix-Trench
a,
*
a
Queensland Institute of Medical Research, RBH Post Office, Herston, Brisbane, QLD 4029, Australia
b
Human Cancer Genetics Unit, Department of Biological Sciences, Brunel University, Uxbridge, Middlesex UB8 3PH, UK
c
Centre for Medical Genetics, Department of Cytogenetics and Molecular Genetics, Women’s and Children’s Hospital, North Adelaide, SA 5006, Australia
Received 2 May 2002; received in revised form 18 September 2002; accepted 2 October 2002
Abstract
Loss of heterozygosity (LOH) of the short arm of chromosome 8 occurs frequently in breast tumors. Finemapping of the smallest regions of overlap of the deletions indicates that multiple tumor suppressor genesmay be located in this region. We have performed microcell-mediated chromosome transfer of chromo-some 8 into two breast cancer cell lines, 21MT-1 and T-47D. Twenty-two of the resulting hybrids werecharacterized extensively with chromosome 8 microsatellite markers and a subset were assayed forgrowth in vitro and soft agar clonicity. There was no evidence in any of the hybrids for suppression ofgrowth or clonicity that could be attributed to the presence of particular regions of chromosome 8; how-ever, none of the 22 hybrids examined had taken up all of the donor chromosome 8, and in fact there werethree regions that contained only one allele of the markers genotyped in all 22 hybrids. These results areconsistent with the presence of suppressor genes on the short arm of chromosome 8 causing strong growthsuppression that is incompatible with growth in vitro; that is, multiple suppressor genes may exist on the
Breast cancer, the most common malignancy in womenliving in the developed world, is poorly understood at themolecular level. Several genes have been identified that, whenmutated in the germline of an individual, can predispose tobreast cancer. In addition, there is evidence from karyo-typic, loss of heterozygosity (LOH), and comparative ge-nomic hybridization (CGH) studies that many somaticevents occur during the transformation of a normal mam-mary precursor cell into a breast tumor. As with other can-cers, it is hypothesized that these somatic events target tu-mor suppressor genes, oncogenes, and genes involved inDNA repair and apoptosis. One of the most common targetsof LOH in breast tumors is the short arm of chromosome 8.This region also undergoes LOH in a wide range of other tu-
mors, such as ovarian, prostate, hepatocellular, bladder, gas-tric, and squamous cell and nonsmall cell lung carcinomas
[1–6]. Deletion mapping suggests that 8p22
�
p23 is the targetof LOH in many of these tumor types, and homozygous de-letions have been identified in oral, pancreatic, head andneck, and prostate cancer at 8p23.3, 8p22, 8p21, and 8p12[2,7–12]. Several candidate tumor suppressor genes havebeen identified on the short arm of chromosome 8, includ-ing
BNIP3L
,
FEZ1/LZTS1
,
DLC1
,
PRLTS
, and
N33
, butnone of them have been found to be frequently inactivatedin breast tumors by either mutation or hypermethylation.
Positional cloning efforts are therefore underway to iden-tify the tumor suppressor gene or genes inactivated by LOHon 8p. In the absence of linkage data, identification of so-matically inactivated tumor suppressor genes is challenging,in part because LOH analysis seldom defines the location ofthese genes to the point where gene identification can be fo-cused with confidence. A complementary strategy is to usemonochromosome transfer of the chromosome of interestinto cancer cell lines, to seek functional evidence for the lo-
P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112
101
cation of putative tumor suppressor genes before attemptinggene identification [13]. This approach can have the addedbenefit that spontaneous deletions in the donor chromosomeoften occur, providing subchromosomal mapping data. Trans-fer of chromosomes 3, 6, 11, 16, and 17 into breast cancercell lines has provided evidence for telomerase repressor,tumor suppressor, metastasis suppressor, or senescence geneson these chromosomes [14–19]; however, the only publishedreport of transfer of chromosome 8 into a breast cancer cell lineused it as a control for the assay of telomerase repression bychromosome 3 and did not evaluate the chromosome 8 hybridsin any detail [20]. The existence of suppressor genes followingmonochromosome transfer is implied by the consistent loss ofcommon eliminated regions (CER) [21,22], and this approachhas been used to identify the
PTEN
gene as the target of chro-mosome 10 deletions in melanoma [23].
About a third of breast tumors have cytogenetic abnor-malities of 8p, many of which result in a net loss of thischromosomal arm [24]. CGH analysis has shown that a de-letion of 8p is one of the most common findings in breastcarcinomas (with reported frequencies of up to 44%),along with gain of 8q and copy number aberrations onmany other chromosomes [25–28]. Similar frequencies of8p deletion have been observed in LOH studies [29,30],with rather higher frequencies in local recurrences, moder-ately differentiated tumors, and
BRCA2
tumors [31–33].Studies in which tumor cells are isolated by microdissec-tion have reported 8p LOH in up to 86% of breast carcino-mas [32,34]. Definition of the critical region is difficultbecause of the relatively small numbers of tumors andmarkers that have usually been examined, but most studiesindicate that the smallest region of overlap (SRO) is within8p12
�
p23 [35–39]. In addition, Wang et al. [40] used 18polymorphic markers from the short arm of chromosome 8to examine 65 cases of ductal carcinoma in situ (DCIS)and defined a SRO at 8p23. Another study of 18 markersin 144 primary invasive breast carcinomas reported thatthe SRO was at 8p22 with another region at 8p12
�
p21[41], which was also reported by Seitz et al. [37] and bySuzuki et al. [42]. In addition, Dahiya et al. [43] identifieddistinct regions of LOH at 8p12 and 8p11 (as well as8q11
�
q12). In summary, the most detailed studies ofLOH in breast cancer indicate that there are commonly de-leted regions at 8p23, 8p22, and 8p12
�
p22.Most studies of 8p LOH in breast carcinomas have re-
ported that deletions on 8p occur in ductal as well as lobu-lar carcinomas [28,35,36]. In an analysis of LOH in 399premalignant breast lesions, O’Connell et al. [44] reportedthat
�
20% of DCIS lesions showed LOH on 8p. Allelicloss at 8p22 is a significant negative prognostic factor forthe survival of certain clinical groups of patients withbreast cancer [33,45]. Both familial and sporadic breastcarcinomas appear to undergo deletions on 8p [29,30,33].There is also some evidence for a breast cancer suscepti-bility gene at 8p12
�
p21 [37,38,46], but this was not con-firmed in a recent study of 31 breast cancer families [47].
Functional evidence for a suppressor gene on chromo-some 8 comes from studies using microcell-mediated mono-chromosome transfer into prostate cancer cell lines which in-dicated that a metastasis suppressor is located at 8p21
�
p12between NEFL and D8S137 [48]. These markers are locatedwithin one of the commonly deleted regions in breast cancer[41]. Transfer of chromosome 8 into colorectal cancer celllines revealed that it carried a tumor suppressor gene [49,50]. The critical region defined for colorectal cancer sup-pression is at 8p22
�
p23, which encompasses two of thecommon regions of LOH in breast cancer [40,41]; however,whether the same genes are targeted in these and other tumortypes that undergo LOH will not be clear until the putativesuppressor genes have been identified. In the present study,we have transferred chromosome 8 into two breast cancercell lines to define the regions carrying putative breast cancersuppression genes.
2. Materials and methods
2.1. Cell lines and culture conditions
Two breast cancer cell lines, 21MT-1 (a gift from V. Band,Dana Farber Institute [51,52] and T-47D (American Type Cul-ture Collection, ATCC, Manassas, VA, USA) were used as re-cipients in the chromosome transfer experiments. 21MT-1,derived from a pleural effusion of a 36-year-old woman withmetastatic breast cancer, was cultured in modified Eagle me-dium-
�
supplemented with 10% fetal bovine serum (FBS),10 mM HEPES, 50
�
g/mL gentamycin, 0.1 mM nonessentialamino acids, 2 mM
L
-glutamine, 1 mM sodium pyruvate,1
�
g/mL insulin, 2.8
�
M hydrocortisone and 12.5 ng/mL epi-dermal growth factor [51]. The T-47D cell line, isolated from apleural effusion of a woman with infiltrating ductal carcinomaof the breast, was obtained from the European Collection ofCell Cultures (ECACC, Salisbury, UK). This cell line wasmaintained in Dulbecco’s modified Eagle medium supple-mented with 10% FBS and 2 mM
L
-glutamine. Mouse A9 cellscontaining a normal human chromosome 8 (A9-Hytk8) orchromosome 20 (A9-Hytk20) tagged with the hygromycin(hyg)-resistance gene were used as donor cells [53]. These cellswere maintained in Dulbecco’s modified Eagle medium con-taining 10% FBS and 400 U/mL hygromycin B.
2.2. Microcell-mediated monochromosome transfer and generation of control transfectant cell line
Microcell-mediated chromosome transfer was performedas described previously [20]. A9-Hytk8 and A9-Hytk20 cellswere treated with 0.1 and 0.075
�
g/mL deacetyl methylcolchi-cine, respectively, to induce micronucleation. After fusionof microcells to recipient cells, hygromycin-resistant (hyg
R
)microcell hybrids were selected in medium containing 400U/mL (21MT-1) or 200 U/mL (T-47D) hygromycin B. Col-onies were examined under the microscope for growth po-tential and were isolated and expanded to mass culture forgenotyping and functional assays. Transfer of chromosome
102
P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112
20 was performed to generate control cell lines; in addition,a 21MT-1 control cell line was generated by transfection ofthe neomycin (neo) acetyltransferase gene (
neo
R
), which con-fers resistance to neomycin. pcDNA3.1 (Invitrogen, Carlsbad,CA, USA) DNA was transfected into 21MT-1 cells (2
�
10
6
)by electroporation. After 24 hours, selection was started by theaddition of 200
�
g/mL G418, and 22 days later a neo-resis-tant colony was isolated. The presence of the hyg
R
gene inthe 21MT-1/8
Hytk
and T-47D/8
Hytk
hybrid cells and the neo
R
gene in the control transfectants was confirmed by poly-merase chain reaction (PCR) amplification [54].
2.3. Cytogenetic analyses of hybrids and chromosome painting
Metaphase spreads were obtained using standard cytoge-netic methods. A minimum of 10 metaphases was counted andthree GTL-banded karyotypes (G-bands by trypsin with Leish-man stain) were produced for each line. Fluorescence in situhybridization (FISH) using a digoxygenin-labeled (DIG-labeled) chromosome 8 painting probe was performed ac-cording to the supplier’s protocol (Vysis, Downers Grove,IL, USA). Slides were mounted for analysis in antifadebuffer containing 4
�
,6-diamidino-2-phenylindole (DAPI) forchromosome identification and propidium iodide as coun-terstain. Slides were viewed using an Olympus AX70 fluo-rescence microscope. Seven to 40 metaphases were scored foreach line, depending on the complexity of the FISH result.Images were captured using the Cytovision ChromoScanimage collection and enhancement system (Applied Imag-ing Corporation, Santa Clara, CA, USA). The FISH signalsand the DAPI banding pattern were merged for figure prep-aration.
2.4. In vitro growth and soft agar cloning assays
Growth rates were determined by plating 1
�
10
4
cells insix 60-mm dishes in medium containing 2% fetal calf serum(FCS); the cells were incubated for 14 days before counting.Doubling times were calculated for each replicate count ac-cording to the formula of Hayflick [55]. For the cell clonic-ity assay, T-47D cells (1
�
10
4
) were plated in a mixture of0.35% agar and 1
�
fully supplemented RPMI-1640 me-dium over a fully supplemented 0.5% agar/1
�
RPMI-1640base. Cells were incubated at 37
�
C in 5% CO
2
for 2 weeksto allow colony formation. Each soft agar cloning experi-ment was performed in triplicate.
2.5. Genotyping of chromosome 8 and 20 microsatellite markers
DNA was extracted from cell lines as described by Milleret al. [56]. Chromosome 8 was genotyped in the donor cellline and the two recipient cell lines with a panel of 57 mic-rosatellite markers from 8p and 14 from 8q to establish theirinformativeness. The markers were selected from the Ge-nome Database (http://www.gdb.org/) and the relative orderof the markers was established from the Celera database
(http://www.celera.com/). The relative order of the markerswas generated through use of the Celera (Rockville, MD,USA) Discovery System and Celera’s associated databases.Forty-two 8p and seven 8q markers were informative in oneor both sets of hygromycin-resistant hybrids (21MT-1/8
Hytk
andT-47D/8
Hytk
) and genotyped in all the relevant hybrids, aswere three chromosome 20 markers (D20S94, D20S162, andD20S206) in the 21MT-1/20
Hytk
and T-47D/20
Hytk
hybrids.The PCR primers were purchased from Research Genetics(Huntsville, AL, USA), Gibco BRL (Rockville, MD, USA),Genset Pacific (Lismore, NSW, Australia), or GeneWorks(Adelaide, SA, Australia) using primer sequences obtainedfrom the Human Genome Database. Standard PCR amplifi-cation was performed in a total volume of 10
�
L using 50ng genomic template DNA incorporating [
33
P]dATP. Mu-rine DNA template was included as a control.
2.6. Statistics
Standard errors for doubling times were calculated basedon the formulae by Hayflick [55] and using the variance ofthe cell counts among the three replicates. The formula cal-culated for the variance of doubling time was
where
t
is the incubation time;
N
is the final cell count;
X
0
isthe initial cell count; and
c
is a constant (
c
�
1/log 2). Natu-ral logarithms were used. Soft agar cloning results were an-alyzed with the
t
-test.
3. Results
3.1. Generation of hybrid and control cell lines
Normal hygromycin-resistant chromosomes 8 and 20were transferred from A9 donor hybrids to 21MT-1 and T-47Dcells by microcell fusion. The 21MT-1 and T-47D cancercell lines were chosen because they contain only one alleleof all 8p microsatellite markers examined, indicating thatthey had undergone LOH in this region. Cytogenetic evi-dence showed that 21MT-1 appeared to have only one copyof 8p, but T-47D appeared to have two copies of 8p, sug-gesting that reduplication had occurred after the LOH.
Eight T-47D/8
Hytk
clones, generated from two transferexperiments, were picked 47–83 days after fusion, and 11T-47D/20
Hytk
clones, generated from one transfer experiment,were picked 72 days after fusion. Twenty-nine 21MT-1/8
Hytk
clones, generated from two transfer experiments, werepicked
�
49 days after fusion, and fourteen 21MT-1/20
Hytk
clones, generated from one transfer experiment, werepicked
�
37 days after fusion. Growth of all the hybrids ap-peared comparable, except that the initial growth of the21MT-1/8 hybrids was slower than that of the 21MT-1/20hybrids (taking longer to reach a size suitable for picking).A 21MT-1
neo
control cell line was generated by the transfection
t2
c2---- Var N( )
N 2------------------
Var X0( )X0
2--------------------+
logN logX0+( )4 , ⁄
P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112
103
of pcDNA3.1 into 21MT-1 cells. Confirmation of the resis-tance gene transfer was obtained by PCR using primers di-rected at the selectable marker (hyg) in all the hybrid celllines (data not shown), and primers directed to the neomy-cin-resistance gene in the neo
R
transfected cell line.
3.2. Genotyping of the 21MT-1/8
Hytk
and T-47D/8
Hytk
hybrids
Seventeen 21MT-1/8
Hytk
, five T-47D/8
Hytk
, two 21MT-1/20
Hytk
, and two T-47D/20
Hytk
hybrids were used for geno-typing. Successful transfer of chromosomes 8 and 20 wasconfirmed by analysis of chromosome 8 (Table 1) and 20(data not shown) microsatellite markers. The A9/8
Hytk
donorcell line showed one allele at every chromosome 8 markertested. Genotyping of the 21MT-1 and T-47D cells with all57 chromosome 8p and 14 chromosome 8q markers revealedthat both recipient cell lines had only a single allele at each8p locus; however, several 8q loci displayed two alleles. Thefollowing 15 markers were uninformative for the 21MT-1 andT-47D cell lines and therefore not used to genotype the
The remaining 42 chromosome 8p microsatellite markerswere used to characterize the 21MT-1/8
Hytk
and T-47D/8
Hytk
hybrids. All of the chromosome 8 hybrids except one,21MT-1/8/2-19, showed the presence of a donated allelefrom A9/8
Hytk
at one or more loci. There was a complex pat-tern of markers in each of the hybrids (Table 1). Out of 22hybrids examined, 16 had a unique chromosome 8 geno-type. Hybrids 21MT-1/8/2-12
Hytk
, 21MT-1/8/2-21
Hytk
, and21MT-1/8/2-24
Hytk
, were identical to one another, as wereT-47D/8/1-1
Hytk
, T-47D/8/2-4
Hytk, and T-47D/8/2-8Hytk. Therewere four loci on 8p (D8S264, D8S1107, D8S1715, andD8S1048) that displayed only one allele in every hybrid, de-spite the fact that 16 of 22 hybrids had a unique chromosome 8genotype. These loci were clustered into three commoneliminated regions: CER 1 at 8p23 (D8S264), CER 2 at8p22�p23 (D8S1107), and CER 3 at 8p21 (D8S1048 andD8S1715). In addition, there were seven loci in these threeregions that displayed only one allele in one set of hybridsand were uninformative in the other set. These seven wereD8S201 (CER 1), D8S1759 (CER 2), D8S1130 (CER 2),D8S1109 (CER 2), D8S133, D8S137, and D8S282 (CER 3).The full extent of these deleted regions could not be evaluatedbecause of lack of informative markers, but their largest es-timated locations are telomere–4.249 Mb (CER 1), 11.626–13.207 Mb (CER 2), and 18.495–28.136 Mb (CER 3). Incontrast to the instability revealed by analysis of chromosome8 markers, there was no evidence from the three microsatel-lite markers tested that deletions of chromosome 20 had oc-curred in the 21MT-1/20Hytk and T-47D/20Hytk hybrids (datanot shown).
In all but one chromosome 8 hybrid cell line (21MT-1/8/2-23Hytk), the single allele present at most of the 8p locinoted above was derived from the recipient cell line (21MT-1or T-47D) but the donor allele had been deleted. In 21MT-1/8/2-23Hytk, however, the donor allele at most loci was clearlypresent but the recipient allele had either been completelydeleted or was very faint and presumably present in only asmall proportion of cells (Table 1). This was true for all theloci in the regions described above, although two alleleswere clearly present at some loci outside of these regions,and three alleles for the 8q markers that were heterozygousin the recipient cell line. None of the informative markersamplified the murine DNA used as a control, ruling out thepossibility that the products observed were derived fromcontaminating murine DNA.
3.3. Cytogenetic analysis
Karyotyping and chromosome 8 painting was performedon the cell lines 21MT-1 and 21MT-1/8/2-1Hytk, 21MT-1/8/2-9Hytk, 21MT-1/8/2-11Hytk, 21MT-1/8/2-12Hytk, and 21MT-1/8/2-23Hytk. The lines were predominantly hyperdiploid, with�58–62 chromosomes per cell. The parental line, 21MT-1,had one normal copy of chromosome 8 plus a marker chro-mosome that appeared to contain the long arm of chromo-some 8 (Fig. 1a). 21MT-1/8/2-1Hytk showed the same chro-mosome-8-containing chromosomes but also an additionalmetacentric chromosome derived from chromosome 8 (Fig.1b). 21MT-1/8/2-9Hytk was similar to 21MT-1 except for theaddition of a small metacentric chromosome derived fromchromosome 8. 21MT-1/8/2-11Hytk resembled 21MT-1, withno apparent additional material from chromosome 8. 21MT-1/8/2-12Hytk was similar to 21MT-1 except for the additionof a metacentric chromosome derived from chromosome 8(Fig. 1c). 21MT-1/8/2-23Hytk resembled 21MT-1 in that mostcells had one normal chromosome 8 and a marker chromo-some that appeared to contain the long arm of chromosome8, but 1 in 15 cells had a second normal copy of chromosome8. Karyotyping and chromosome 8 painting of T-47D cellsshowed that they were hyperdiploid with 62–64 chromo-somes per cell. The majority had two copies of chromosome8, as well as two copies of a large metacentric chromosome,one end of which was derived from chromosome 8 (Fig.1d). The T-47D/8/1-1Hytk, T-47D/8/2-4Hytk, and T-47D/8/2-8Hytk lines all contained one or two normal copies of chro-mosome 8, one to four copies of a large metacentric withone arm painted, and a large metacentric with both armspainted (Fig. 1e). The T-47D/8/2-1Hytk and T-47D/8/2-7Hytk
lines were similar to the T-47D cells except that, in additionto one normal copy of chromosome 8, there was an 8q�marker chromosome.
3.4. In vitro characterization of hybrids
Cell morphology did not appear to be dramatically al-tered in any of the hybrids compared with the parental cell
104 P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112
Tab
le 1
Mic
rosa
telli
te m
arke
r an
alys
is in
21M
T-1
/8hy
g an
d T
-47D
/8hy
g hy
brid
s ce
ll lin
es
STR
Mb
21M
T-1
alle
les
(no.
)
21M
T-1
/8H
ytk hy
brid
sT
-47D
alle
les
(no.
)
T-4
7D/8
Hyt
k hyb
rids
CE
Rs
1-2
1-3
2-1
2-5
2-7
2-8
2-9
2-11
2-12
2-18
2-19
2-20
2-21
2-22
2-23
2-24
2-25
1.1
2.1
2.4
2.7
2.8
D8S
264
2.75
1r
rr
rr
rr
rr
rr
rr
rd
rr
1r
rr
rr
CE
R 1
D8S
201
3.75
71
rr
rr
rr
rr
rr
rr
rr
dr
r1
nini
nini
niC
ER
1D
8S18
244.
249
1B
BB
rB
rB
rB
Br
rB
rd
Br
1B
BB
BB
D8S
1781
4.27
51
BB
Br
Br
Br
BB
rr
Br
dB
r1
BB
BB
BD
8S26
24.
373
1B
Br
rB
rB
rB
Br
rB
rd
Br
1ni
nini
nini
D8S
1798
5.79
61
BB
BB
BB
rr
Bnd
rr
Br
dB
r1
Br
Br
BD
8S30
76.
702
1B
BB
ndB
ndr
rB
Br
rB
rd
Br
1nd
rB
BB
D8S
277
7.21
31
nini
nini
nini
nini
nini
nini
nini
nini
ni1
Br
BB
BD
8S14
699.
367
1B
Br
rB
rr
rB
Br
rB
rd
Br
1B
rB
BB
D8S
503
9.51
11
nini
nini
nini
nini
nini
nini
nini
nini
ni1
Br
BB
BD
8S51
69.
688
1B
Br
rB
rr
rB
Br
rB
rd
Br
1ni
nini
nini
D8S
1721
10.4
151
Br
rB
rB
rB
Br
rr
Br
dB
r1
Br
BB
BD
8S52
010
.832
1B
Br
rB
BB
rB
Br
rB
rd
Br
1ni
nini
nini
D8S
550
11.1
561
BB
Br
BB
Br
BB
rr
Br
dB
r1
Br
BB
BD
8S16
9511
.626
1B
Br
rB
rB
rB
Br
rB
rd
Br
1B
rB
BB
D8S
1759
11.7
541
rr
rr
rr
rr
rr
rr
rr
dr
r1
nini
nini
niC
ER
2D
8S11
3012
.105
1r
rr
rr
rr
rr
rr
rr
rd
rr
1ni
nini
nini
CE
R 2
D8S
1109
12.9
751
rr
rr
rr
rr
rr
rr
rr
dr
r1
nini
nini
niC
ER
2D
8S11
0713
.042
1r
rr
rr
rr
rr
rr
rr
rd
rr
1r
rr
rr
CE
R 2
D8S
1790
13.2
071
BB
rr
BB
rr
BB
rr
Br
BB
B1
nini
nini
niD
8S18
2714
.96
1B
Br
rB
BB
rB
Br
rB
BB
BB
1B
BB
BB
D8S
1992
15.6
511
nini
nini
nini
nini
nini
nini
nini
nini
ni1
BB
BB
BD
8S54
915
.792
1ni
nini
nini
nini
nini
nini
nini
nini
nini
1B
BB
BB
D8S
1991
15.9
121
BB
rr
BB
Br
BB
rr
BB
dB
B1
BB
BB
BD
8S11
3516
.207
1B
Br
rB
BB
rB
Br
rB
Bd
BB
1B
BB
BB
D8S
254
16.7
511
BB
rr
BB
Br
BB
rr
BB
dB
B1
BB
BB
BD
8S11
4518
.495
1B
Br
rB
rB
rB
Br
rB
rd
BB
1B
BB
BB
D8S
1715
19.9
291
rr
rr
rr
rr
rr
rr
rr
dr
r1
rr
rr
rC
ER
3D
8S28
221
.557
1r
rr
rr
rr
rr
rr
rr
rd
rr
1ni
nini
nini
CE
R 3
D8S
133
22.1
031
rr
rr
rr
rr
rr
rr
rr
dr
r1
nini
nini
niC
ER
3D
8S10
4826
.95
1r
rr
rr
rr
rr
rr
rr
rd
rr
1r
rr
rr
CE
R 3
D8S
137
27.8
1ni
nini
nini
nini
nini
nini
nini
nini
nini
1r
rr
rr
CE
R 3
D8S
1820
28.1
361
BB
rr
Br
Br
BB
rr
Br
dB
r1
BB
BB
BD
8S49
932
.279
1B
BB
rB
rr
rB
Br
rB
rd
Br
1B
BB
BB
D8S
278
32.6
861
nini
nini
nini
nini
nini
nini
nini
nini
ni1
BB
BB
BD
8S50
534
.581
1ni
nini
nini
nini
nini
nini
nini
nini
nini
1B
rB
BB
D8S
379
34.7
471
rr
rr
rr
rr
rr
rr
rr
dr
r1
nini
nini
niD
8S87
35.4
31
rr
ndr
rr
rr
rr
rr
rr
dr
r1
Br
BB
BD
8S11
2135
.931
1r
rr
rr
rr
rr
rr
rr
rd
rr
1ni
nini
nini
D8S
255
39.8
541
rB
rr
BB
rr
BB
rr
Br
BB
r1
nini
nini
niA
NK
141
.477
1r
rr
rr
rr
rr
rr
rr
rd
rr
1B
BB
BB
(Con
tinu
ed)
P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112 105
Tab
le 1
(Con
tinu
ed)
STR
Mb
21M
T-1
alle
les
(no.
)
21M
T-1
/8H
ytk h
ybri
dsT
-47D
alle
les
(no.
)
T-4
7D/8
Hyt
k hyb
rids
CE
Rs
1-2
1-3
2-1
2-5
2-7
2-8
2-9
2-11
2-12
2-18
2-19
2-20
2-21
2-22
2-23
2-24
2-25
1.1
2.1
2.4
2.7
2.8
D8S
1833
50.6
602
nini
nini
nini
nini
nini
nini
nini
nini
ni1
Br
BB
BD
8S16
654
.985
2B
Br
rB
rnd
rB
Br
rB
rB
BB
1B
rB
BB
D8S
1117
67.3
772
BB
BB
Bnd
Br
BB
rr
BB
BB
r2
nini
nini
niD
8S28
381
.229
1B
BB
rB
Br
rB
Br
rB
rB
BB
1ni
nini
nini
D8S
1047
108.
927
2B
Br
rB
BB
rB
Br
rB
BB
BB
2B
BB
BB
D8S
1022
112.
320
2ni
nini
nini
nini
nini
nini
nini
nini
nini
2B
BB
BB
D8S
342
122.
082
1B
Br
rB
BB
rB
Br
rB
rB
BB
1B
BB
BB
D8S
568
125.
132
2B
Br
rB
rr
BB
rr
BB
Br
BB
1ni
nini
nini
Ital
ic a
cros
s en
tire
row
indi
cate
s re
gion
s w
here
onl
y on
e al
lele
was
pre
sent
in e
very
info
rmat
ive
hybr
id. T
he th
ree
CE
Rs
iden
tifie
d ar
e sh
own
in th
e la
st c
olum
n.A
bbre
viat
ions
: B, b
oth
dono
r an
d re
cipi
ent a
llele
s pr
esen
t (bo
ldfa
ce);
CE
R, c
omm
on e
limin
ated
reg
ion;
d, o
nly
dono
r al
lele
pre
sent
; nd,
not
don
e; M
b, m
egab
ase;
ni,
not i
nfor
mat
ive
(don
or a
nd r
ecip
ient
al-
lele
s in
dist
ingu
isha
ble
by s
ize)
; r, o
nly
reci
pien
t alle
le p
rese
nt; S
TR
, sho
rt ta
ndem
rep
eat m
arke
r.
lines. In vitro growth assays were performed with the21MT-1 parental cell line and six chromosome 8-containinghybrids compared with three control cell lines (Table 2).Four of the 21MT-1/8Hytk hybrids had significantly longerdoubling times than the 21MT-1 cell line (21MT-1/8/1-2Hytk,21MT-1/8/2-1Hytk, 21MT-1/8/2-12Hytk, and 21MT-1/8/2-23Hytk),as did two of the control cell lines (21MT-1/20/1-3Hytk and21MT-1/neo1). The doubling times of five of the T-47D/8Hytk
hybrids (T-47D/8Hytk/1.1, 2.1, 2.4, 2.7, and 2.8) were evalu-ated and compared with those of the parental cell line and oftwo T-47D/20Hytk control cell lines. T-47D/8/1-1Hytk andT-47D/8/2-7Hytk cells had a significantly higher doubling time(74.2 and 72.3 hours, respectively) compared with the T-47Dparental cells (50.8 hours).
21MT-1 cells do not clone in soft agar, so the soft agar clon-ing assay could be performed only with the T-47D hybrids(T-47D/8Hytk 1.1, 2.1, 2.4, 2.7, and 2.8). One of the T-47D/8Hytk
hybrids, T-47D/8/2-4, did not form colonies in soft agar,and two of the other chromosome 8 containing hybrids,T-47D/8/1-1Hytk and T-47D/8/2-1Hytk had significantly re-duced colony formation compared with the parental cell line(62 and 35%, respectively), as did one of the chromosome 20-containing control cell lines, T-47D/20/1-5 (70%) (Table 2).
4. Discussion
LOH and CGH analyses of breast tumors have providedevidence for at least three tumor suppressor genes on theshort arm of chromosome 8 [37,40–42].
We performed microcell-mediated chromosome transferof chromosome 8 into two breast cancer cell lines, 21MT-1and T-47D, chosen because they contain only one allele ofall 8p microsatellite markers examined, indicating LOH inthis region. Cytogenetic evidence showed that 21MT-1 ap-peared to have only one copy of 8p but T47-D appeared tohave two copies of 8p, suggesting that reduplication oc-curred after the LOH. Thirty-seven hybrids were obtained intotal, of which 22 were characterized by genotyping, 11were assayed for in vitro growth, and 5 (all the T-47D/8 hy-brids) for soft agar clonicity (Table 2). In comparison withcontrols into which chromosome 20 or the neomycin-resis-tance gene had been transferred, no significant changes inphenotype were observed in the 21MT-1/8 hybrids attribut-able to the presence of chromosome 8. Two of the T-47D/8hybrids had a significantly longer doubling time than theT-47D cell line (74.2 and 72.3 hours, compared with 50.8hours), but this could not be attributed to the presence ofparticular regions of 8p in the hybrids. One of the T-47D/20control cell lines had a doubling time of 77.6 hours, and sothe slower growth of the T-47D/8/1-1 and T-47D/8/2-7 hy-brids may represent clonal variation in the T-47D cell line.Three of the T-47D/8 cell lines and one of the controls hadsignificantly reduced colony formation in soft agar, includ-ing T-47D/8/2-4, which did not clone at all. Extensive geno-typing did not reveal the presence of any part of the donated
106 P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112
Fig. 1. Chromosome 8 painting showing marker chromosomes in the recipient and hybrid cell lines. (a) 21MT-1Hytk. Short arrow indicates apparently normalcopy of chromosome 8, long arrow indicates marker chromosome containing the long arm of chromosome 8. (b) 21MT-1/8/2-1Hytk. Short arrow indicatesapparently normal copy of chromosome 8, long arrow indicates the same marker chromosome as seen in 21MT-1, and arrowhead indicates additional chro-mosome. (c) 21MT-1/8/2-12Hytk. Long arrow indicates the same marker chromosome as seen in 21MT-1 and short arrows indicate an apparently normal copyof chromosome 8, as well as an additional chromosome. (d) T-47DHytk. Short arrows indicate apparently normal copies of chromosome 8 and long arrowsindicate metacentric markers, one end of which is derived from chromosome 8. (e) T-47D/8/1-1Hytk. Short arrows indicate apparently normal copies of chro-mosome 8, long arrows indicate metacentric markers, one end of which is derived from chromosome 8, and arrowhead indicates additional metacentricmarker chromosome painted at both ends.
P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112 107
chromosome 8 in the T-47D/8/2-4 hybrid that was not alsopresent in the T-47D/8/2-8 hybrid, which did not show sup-pression of soft agar clonicity. Given the reduction in softagar clonicity in one of the controls, it is likely that our re-sults are a reflection of clonal variation in the parental cellline. No in vivo tumorigenicity assays were performed, be-cause of the long latency and poor tumor-forming ability ofthe T-47D cell line in mice (more than 3 months).
Successful chromosome transfer was indicated by PCRof the hygromycin resistance gene, karyotypic evidence andanalysis of chromosome 8 microsatellite markers. With theexception of 21MT-1/8/2-19 (which clearly contained thehygR gene and must therefore have taken up a small part ofthe donor chromosome), all the hybrids contained the donorallele at one or more of the 49 informative loci examined.Analysis of the genotyping results, however, revealed tworemarkable features of these hybrids. First, no hybrid hadtaken up the entire donor chromosome 8. Multiple deletionswere present within the short arm of the donated chromo-some 8, but certain loci appeared to have been targeted bythese deletions. Despite the fact that 16 of the 22 hybrids ex-amined had unique marker patterns and therefore werelikely to have arisen independently, all of them had in com-mon the presence of a single allele at four loci (D8S264,D8S1107, D8S1715, and D8S1048) that mapped to threecommonly eliminated regions on 8p (CER 1–3; Table 1).
Second, in 21 of the hybrids only the recipient allele waspresent; in 21MT-1/8/2-23Hytk, however, the recipient allelehad been almost completely deleted but the donor allele waspresent. Where the recipient allele was detected, it was usu-ally much fainter on the autoradiograph than the donor al-lele, indicating that it was not present in all cells.
These genotyping data are consistent with the karyotypicevidence. Additional chromosome material detectable withchromosome 8 paint was noted in the 21MT-1/8/2-1Hytk,21MT-1/8/2-9Hytk, and 21MT-1/8/2-12Hytk hybrids and in allthe T-47D/8Hytk hybrids. No additional material was notedin 21MT-1/8/2-11Hytk, which appeared to have taken uponly a small part of the donor chromosome 8, nor in 21MT-1/8/2-23Hytk. 21MT-1/8/2-23Hytk appeared very similar to therecipient cell line, which is consistent with genotypic evi-dence of it having lost the recipient chromosome 8 andtaken up the donor copy instead. An additional chromosome8 was noted in 1 out of 15 cells examined, presumably de-rived from the recipient, consistent with the presence of therecipient alleles that was just visible at some loci.
These results are consistent with the presence of suppres-sor genes on the short arm of chromosome 8 that causestrong growth suppression, incompatible with growth invitro. This suggests that the hybrids were all revertants thatarose after spontaneous deletion of the donor chromosomeoccurred (or, in the case of 21MT-1/8/2-23, after the recipi-ent chromosome was deleted). In this regard, note that theinitial growth of the 21MT-1/8 hybrids was slower than thatof the 21MT-1/20 hybrids (taking longer to reach a size suit-able for picking) and that only 8 T-47D/8 hybrids were ob-tained from two fusion experiments, in contrast to 11 T-47D/20hybrids from a single experiment. Alternatively, there mayhave been selection at the time of microcell fusion, so thatonly clones that took up a deleted version of the donor chro-mosome survived. A third possibility is that the donor cellline contained subclones that carried several different de-leted donor chromosomes and that only hybrids that took upthe deleted donor chromosome survived, once again sug-gesting selection against these regions. If any of these hy-potheses are correct, it is not surprising that none of thechromosome-8 containing hybrids had significantly longerdoubling times or diminished soft agar clonicity comparedwith controls.
Given the tendency for donor chromosomes to fragmentduring microcell-mediated chromosome transfer [14,16–18,20,48,50,57], it is not surprising that in most cases it was thedonor chromosome that appeared to have undergone dele-tion, rather than the recipient. What is more remarkable isthat in one hybrid, 21MT-1/8/2-23Hytk, the reverse happenedand the donor chromosome was retained in preference to therecipient chromosome. This is inconsistent with the Knud-sen hypothesis, which suggests that the recipient alleles ofthe putative suppressor genes are inactivated by mutationand that this could be complemented by the transfer of anormal copy of chromosome 8 from a donor cell line [58].Instead, this is consistent with a mechanism of haploinsuffi-
Table 2In vitro characteristics of recipient cell lines and microcell-fusion derived hybrid cell lines
aMean number of colonies (based on 3 replicates) consisting of greaterthan 20 cells that formed in soft agar per 104 cells plated; standard error(SE) in parentheses.
bMean doubling time (based on 3 replicates) based on 104 cells initially;standard error (SE) in parentheses.
* P 0.05 in comparison to the parental line.
108 P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112
Table 3Overlap between regions of LOH, regions of suppression, homozygous deletions, and CERs on the short arm of chromosome 8
P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112 109
ciency acting at these putative suppressor loci in the 21MT-1cell line. A similar situation has been reported recently in theSyrian hamster cell line BHK-191-5C, in which introductionof a human derivative chromosome 9 was accompanied bythe loss of Syrian hamster chromosome 10 [59]. Accumulat-ing evidence suggests that the Knudsen two-hit mechanism ofinactivation of suppressor genes may not always apply tosomatic tumors, but that instead half the normal levels of thegene might be sufficient to contribute to tumorigenesis [60–64]. If our hypothesis were correct—if these hybrids are re-vertants and that the CERs consistently deleted in all the in-formative hybrids contain in vitro growth suppressor genes—it would suggest that three suppressor genes may be locatedon the short arm of chromosome 8. Only detailed examina-tion of the genes in these CERs will determine if any of themact as suppressor genes and whether they are inactivated by ge-netic or epigenetic mechanisms. Several candidate tumor sup-pressor genes have been identified on the short arm of chromo-some 8, including BNIP3L, FEZ1/LZTS1, DLC-1, PRLTS, andN33; however, only BNIP3L, FEZ1/LZTS1, and DLC-1 liewithin the CERs (at 26.379, 20.254, and 13.131 Mb, respec-tively). There is no convincing evidence at present that any
of these are the critical suppressor genes in the CERs. Wehave recently examined 25 breast tumors, with and without 8pLOH, for BNIP3L mutations but found none [65]. In addi-tion, mutation analysis of DLC-1 has failed to find any mu-tations in colorectal or ovarian tumors [66]; no analysis ofbreast tumors has been conducted to date. FEZ1/LZTS1 isnot expressed in breast cancer cell lines or primary tumors[67] and transfection experiments indicate that it can suppressgrowth of breast cancer cell lines by regulation of mitosis[68]. To date, however, no mutations have been reported inbreast tumors [67], and there is no evidence that FEZ1/LZTS1 is hypermethylated in breast tumors. It is thereforenot clear whether loss of FEZ1/LZTS1 is critical to breastcancer development or, if so, at what frequency.
We looked at the location of the CERs with respect to thecommon regions of LOH reported in the literature. We haveused the order of markers given by the Celera database toredefine the regions of interest on 8p that have been pub-lished previously (Table 3). Notably, CER 3 overlaps thecommon regions of LOH at 8p12�p21 reported by Seitz et al.[37], Suzuki et al. [42], and Yokota et al. [41], as well as theregion identified as capable of suppressing prostate metasta-
Abbreviations: �, span of region indicated; BRCA, breast cancer; CRCSR, colorectal cancer suppressor region; LOH, smallest region of overlap of loss ofheterozygosity; Mb, megabase; OVCA, ovarian cancer; PCSR, prostate cancer suppressor region; STR, short tandem repeat marker.
110 P. Wilson et al. / Cancer Genetics and Cytogenetics 143 (2003) 100–112
sis [48] and two small homozygous deletions identified inprostate carcinoma [8]. These data suggest that the putativein vitro growth suppressor gene or genes that exist on theshort arm of chromosome 8 may operate in more than onetissue type.
Acknowledgments
We gratefully acknowledge Helena Kelly and John Lai fortechnical assistance and The Kathleen Cuningham Founda-tion, the National Health and Medical Research Council ofAustralia, The Wellcome Trust, and the Queensland CancerFund for support. G.C.-T. is supported by a Senior ResearchFellowship from the National Health and Medical ResearchCouncil of Australia.
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