Genetic variability in MCF-7 sublines: Evidence of rapid genomic and RNA expression profile modifications

Equipe Génome et Cancer, UMR 5535, CNRS and EMI 0229, INSERM, Centre de Recherche CRLC Val d'Aurelle, Montpellier, France.
BMC Cancer (Impact Factor: 3.36). 05/2003; 3:13. DOI: 10.1186/1471-2407-3-13
Source: PubMed
ABSTRACT
Both phenotypic and cytogenetic variability have been reported for clones of breast carcinoma cell lines but have not been comprehensively studied. Despite this, cell lines such as MCF-7 cells are extensively used as model systems.
In this work we documented, using CGH and RNA expression profiles, the genetic variability at the genomic and RNA expression levels of MCF-7 cells of different origins. Eight MCF-7 sublines collected from different sources were studied as well as 3 subclones isolated from one of the sublines by limit dilution.
MCF-7 sublines showed important differences in copy number alteration (CNA) profiles. Overall numbers of events ranged from 28 to 41. Involved chromosomal regions varied greatly from a subline to another. A total of 62 chromosomal regions were affected by either gains or losses in the 11 sublines studied. We performed a phylogenetic analysis of CGH profiles using maximum parsimony in order to reconstruct the putative filiation of the 11 MCF-7 sublines. The phylogenetic tree obtained showed that the MCF-7 clade was characterized by a restricted set of 8 CNAs and that the most divergent subline occupied the position closest to the common ancestor. Expression profiles of 8 MCF-7 sublines were analyzed along with those of 19 unrelated breast cancer cell lines using home made cDNA arrays comprising 720 genes. Hierarchical clustering analysis of the expression data showed that 7/8 MCF-7 sublines were grouped forming a cluster while the remaining subline clustered with unrelated breast cancer cell lines. These data thus showed that MCF-7 sublines differed at both the genomic and phenotypic levels.
The analysis of CGH profiles of the parent subline and its three subclones supported the heteroclonal nature of MCF-7 cells. This strongly suggested that the genetic plasticity of MCF-7 cells was related to their intrinsic capacity to generate clonal heterogeneity. We propose that MCF-7, and possibly the breast tumor it was derived from, evolved in a node like pattern, rather than according to a linear progression model. Due to their capacity to undergo rapid genetic changes MCF-7 cells could represent an interesting model for genetic evolution of breast tumors.

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BMC Cancer
Open Access
Research article
Genetic variability in MCF-7 sublines: evidence of rapid genomic
and RNA expression profile modifications
Mélanie Nugoli
1
, Paul Chuchana
1
, Julie Vendrell
1
, Béatrice Orsetti
1
,
Lisa Ursule
1
, Catherine Nguyen
2
, Daniel Birnbaum
3
,
Emmanuel JP Douzery
4
, Pascale Cohen
1,5
and Charles Theillet*
1
Address:
1
Equipe Génome et Cancer, UMR 5535 CNRS and EMI 0229 INSERM Centre de Recherche CRLC Val d'Aurelle, Montpellier, France,
2
Laboratoire TAGC, CIML, Université d'Aix-Marseille II, Marseille, France,
3
INSERM U119 and LBT, Institut Paoli Calmette, 232 blv Ste Marguerite,
13009 Marseille, France,
4
Institut des Sciences de l'Evolution de Montpellier CNRS UMR 5554, Université des Sciences et Techniques du
Languedoc Montpellier II, Montpellier, France and
5
Institut de Biotechnologies et Pharmacologie CNRS UMR 5094, Faculté de Pharmacie
Université Montpellier I, Montpellier, France
Email: Mélanie Nugoli - nugoli@valdorel.fnclcc.fr; Paul Chuchana - chuchana@valdorel.fnclcc.fr; Julie Vendrell - vendrell@valdorel.fnclcc.fr;
Béatrice Orsetti - orsetti@valdorel.fnclcc.fr; Lisa Ursule - ursule@valdorel.fnclcc.fr; Catherine Nguyen - nguyen@ciml.univ-mrs.fr;
Daniel Birnbaum - birnbaum@marseille.inserm.fr; Emmanuel JP Douzery - douzery@isem.isem.univ-montp2.fr;
Pascale Cohen - pascale.cohen@ibph.pharma.univ-montp1.fr; Charles Theillet* - theillet@valdorel.fnclcc.fr
* Corresponding author
Abstract
Background: Both phenotypic and cytogenetic variability have been reported for clones of breast
carcinoma cell lines but have not been comprehensively studied. Despite this, cell lines such as
MCF-7 cells are extensively used as model systems.
Methods: In this work we documented, using CGH and RNA expression profiles, the genetic
variability at the genomic and RNA expression levels of MCF-7 cells of different origins. Eight MCF-
7 sublines collected from different sources were studied as well as 3 subclones isolated from one
of the sublines by limit dilution.
Results: MCF-7 sublines showed important differences in copy number alteration (CNA) profiles.
Overall numbers of events ranged from 28 to 41. Involved chromosomal regions varied greatly
from a subline to another. A total of 62 chromosomal regions were affected by either gains or
losses in the 11 sublines studied. We performed a phylogenetic analysis of CGH profiles using
maximum parsimony in order to reconstruct the putative filiation of the 11 MCF-7 sublines. The
phylogenetic tree obtained showed that the MCF-7 clade was characterized by a restricted set of
8 CNAs and that the most divergent subline occupied the position closest to the common
ancestor. Expression profiles of 8 MCF-7 sublines were analyzed along with those of 19 unrelated
breast cancer cell lines using home made cDNA arrays comprising 720 genes. Hierarchical
clustering analysis of the expression data showed that 7/8 MCF-7 sublines were grouped forming
a cluster while the remaining subline clustered with unrelated breast cancer cell lines. These data
thus showed that MCF-7 sublines differed at both the genomic and phenotypic levels.
Conclusions: The analysis of CGH profiles of the parent subline and its three subclones supported
the heteroclonal nature of MCF-7 cells. This strongly suggested that the genetic plasticity of MCF-
7 cells was related to their intrinsic capacity to generate clonal heterogeneity. We propose that
Published: 24 April 2003
BMC Cancer 2003, 3:13
Received: 6 December 2002
Accepted: 24 April 2003
This article is available from: http://www.biomedcentral.com/1471-2407/3/13
© 2003 Nugoli et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
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MCF-7, and possibly the breast tumor it was derived from, evolved in a node like pattern, rather
than according to a linear progression model. Due to their capacity to undergo rapid genetic
changes MCF-7 cells could represent an interesting model for genetic evolution of breast tumors.
Background
Primary breast tumors are known for their elevated level
of inter-tumor heterogeneity, however, an important
body of data has brought evidence of intra-tumoral heter-
ogeneity as well. Such evidence stems from cytogenetic
studies which have shown that cytogenetically unrelated
clones can be found in breast tumors [1]. These findings
have been interpreted either as the result of genetic insta-
bility following loss of proper mitotic controls [2], or as
the expression of the admixture of multiple genetically
non related cellular clones [3]. Flow cytometry has been
another way to address the question of intratumoral het-
erogeneity, showing that breast tumors correspond to in-
tricate admixture of tumor cells with different DNA
contents (i.e. different ploidies) [4]. These findings were
extended by Bonsing and coworkers [5], who showed that
diploid and aneuploid cells, concurently present in breast
tumors, had a number of genetic anomalies in common.
In fact, all the allelic imbalances observed in the diploid
compartment were found in aneuploid cells. This was a
strong indication of a direct filiation between diploid and
aneuploid cells in breast tumors. Heterogeneity is thus a
major problem in mammary carcinogenesis and has im-
portant clinical implications in terms of prognosis and
therapy.
MCF-7 cells are the most commonly used model of estro-
gen positive breast cancer. This cell line has been original-
ly established in 1973 at the Michigan Cancer Foundation
from a pleural effusion taken from a woman with meta-
static breast cancer [6] and since then MCF-7 cells have
been widely distributed in laboratories throughout the
world resulting in the production of different cellular
stocks. Quite early in the history of MCF-7 cells reports on
clonal variations were made in the literature. Most of the
reported differences concerned phenotypic traits such as
estrogen responsiveness or ability to form tumors in syn-
geneic mice, but karyotypic differences were observed as
well [7–9]. MCF-7 cells presented extensive aneuploidy
with important variations in chromosome numbers rang-
ing from 60 to 140 according to the variant examined.
Other cytogenetic differences concerned the presence or
absence of specific marker chromosomes. While loss of
marker chromosomes seemed a rare event, occurrence of
new aberrations was more common [8]. However, some
doubt remained on the true origin of these differences, as
some MCF-7 sublines corresponded to other cancer cells
of unknown origin [10].
The available data suggested an elevated level of genetic
instability in MCF-7 cells. The observed karyotypic differ-
ences could reflect changes in selective pressure due to dif-
ferent culture conditions. Alternatively, work by Resnicoff
and coworkers [11] showed that, upon fractionation of
MCF-7 cells on a Percoll gradient, it was possible to isolate
six different subpopulations, one of which bore the capac-
ity to regenerate all other cellular populations. These data
suggested that MCF-7 cells contain a fraction of stem cells
able to generate clonal variability. This was proposed as
an explanation for the heterogeneity of this cell line and
as a model for breast tumor heterogeneity.
In a previous work [12] we analyzed by Comparative Ge-
nomic Hybridization (CGH) two sublines of MCF-7 cells
which showed surprisingly different genomic profiles.
Our data were concordant with that reported by Jones and
coworkers [13]. We became interested in: (1) document-
ing the genetic variability, at both genomic and RNA ex-
pression levels, that exists among different MCF-7
sublines of different origins; (2) retracing their evolution-
ary history and unraveling their filiation; (3) addressing
the issue of the cause of this diversity and whether it re-
flected their intrinsic capacity to generate clonal heteroge-
neity or resulted from local changes in culture conditions.
The resulting information should help understand tumor
heterogeneity.
To address these questions we collected 9 cell lines identi-
fied as MCF-7 variants. We also established 3 cell clones
starting from one of the collected sublines. The different
MCF-7 variants were compared at the genetic level using
CGH as well as RNA expression profiling. CGH and RNA
expression profiles were subjected to phylogenetic analy-
ses to determine the degree of filiation between the differ-
ent cell lines studied.
Methods
Cell lines
Eleven variants or sublines of MCF-7 cells were tested in
this study: MCF-7-ATCC, MCF-7-R, MCF-7-O, MCF-7-MF
and MCF-7-MG, MCF-7-MVLN, MCF-7-MVLN-6ms7,
MCF-7-MVLN-6ms8, MCF-7-R-F3, MCF-7-R-D4, MCF-7-
R-G1. MCF-7-ATCC were obtained from Dr A. Pèlegrin
(Cancer Center, Montpellier, France) who purchased it
from ATCC in 1996. Cells were at passage 143 when we
analyzed them. MCF-7-R, MCF-7-O, MCF-7-MF and MCF-
7-MG were all obtained from Dr F. Vignon (INSERM,
Montpellier, France). MCF-7-R were originally obtained
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from Dr Rich (Michigan Cancer Foundation, USA) by Dr
F. Vignon in 1985, analyzed cells were at passage 61 (pas-
sage 0 at the time of arrival of the cells in Dr Vignon's lab-
oratory). MCF-7-O were obtained from Dr Osborne
(Texas Health Science Center, San Antonio) in 1987, ana-
lyzed cells were at passage 62. MCF-7-MF were obtained
from Dr Lippman (Lombardi Cancer Center, Washington
DC, USA) in 1988, analyzed cells were at passage 5. MCF-
7-MG were obtained from Dr Mc Guire (Texas Health Sci-
ence Center, San Antonio) in 1994, analyzed cells were at
passage 338. MCF-7-MVLN were obtained from Drs. J.C.
Nicolas and M. Pons (INSERM, Montpellier, France).
These cells correspond to MCF-7 cells transfected with a
construct containing the Luciferase gene under control of
an ERE sequence. MCF-7-MVLN-6ms7 and MCF-7-
MVLN-6ms8, respectively correspond to two subclones of
MCF-7-MVLN cells which have developped resistance to
Tamoxifene. MCF-7-R-F3, MCF-7-R-D4 and MCF-7-R-G1
correspond to cell clones established in our laboratory by
limit dilution from MCF-7-R cells.
Other breast cancer lines used in this study included Brca-
MZ-01 and Brca-MZ-02, MDA-MB-436 (kindly provided
by Dr. A. Puisieux), BT-20, CAMA-1, HCC 1187, HCC
1428, HCC 1569, HCC 1937, HCC 1954, HCC 2218,
MCF10F, MDA-MB-175, UACC-812 (ATCC, Manassas,
Va.), EFM-19, EFM-192A (DSMZ, Braunschweig, Germa-
ny), KPL-1, SUM 149, SUM-229 (kindly provided by Dr S.
Ethier). The Doxorubicin resistant line was provided to us
by Dr Frederic Pinguet (Canter Center Montpellier). All
cell lines were maintained in DMEM containing 10% FBS
supplemented with L-Glutamine (200 mM, 100X) and
Antibiotic-Antimycotic (100X) GibcoBRL, Life Technolo-
gies, Cergy Pontoise.
DNA and RNA purification
Genomic DNA and total RNA were isolated as previously
described [14]. RNA integrity was controlled by denatur-
ing formaldehyde agarose electrophoresis and checked by
Northern blot, hybridizing the RNA with an oligonucle-
otide probe specific to the 28S rRNA.
Genetic analysis of the different sublines
All the cell lines used in this study have been haplotyped
with a combination of 9 CA repeat microsatellite markers
from the Généthon collection, respectively localized on
chromosomes 1, 6 and 17: D1S2615, D1S2811,
D1S2624, D6S310, D6S401, D6S460, D17S1855,
D17S1865, D17S1604. Primers are described on the
Généthon web site http://www.genlink.wustl.edu/
genethon_frame/. PCR conditions and size analysis of the
products were as described [15].
Comparative Genomic Hybridization
Metaphase preparation, genomic DNA labeling, CGH re-
action and image analysis were as described [16].
Phylogenetic analysis on CGH data
The evolutionary history of cell lineages was reconstructed
in a cladistic framework. Chromosomal bands were con-
sidered as characters, existing under three possible discrete
character states: gain, loss and normal (i.e. no mutation).
Transformations from one character state to another were
equally weighted, and the normal state (i.e. tumor/nor-
mal hybridization ratio = 1) was considered ancestral.
Phylogenetic trees were reconstructed under the maxi-
mum parsimony (MP) criterion, using the following hy-
pothesis [17]: (1) all characters were considered as
independent: i.e. events occurring at one band did not af-
fect events occurring at another band; (2) they were unor-
dered : it was possible to directly change from one state
(either normal, amplified, or deleted) to a second one,
without invoking the third one; (3) they were equally
weighted : each change from one state to another had the
same probability of occurrence. Using the MP approach
has two main advantages: (1) it considered chromosome
bands one by one, and integrated CGH information avail-
able for each of them for all twelve cell lineages simulta-
neously ; (2) it allowed to trace a posteriori chromosomal
events that characterize the different groups of cell lineag-
es evidenced on the most parsimonious trees and to iden-
tify diagnostic events. All analyses were conducted with
PAUP* [18], version 4 beta 8, with heuristic MP searches
based on 1000 random addition of cell lineages, with tree
bisection-reconnection (TBR) branch swapping, and ac-
celerated transformation (ACCTRAN) optimization of
character-states. To trace the character-state changes along
the phylogenetic trees, we used the program MacClade
[19], version 3.04. In order to evaluate whether CGH data
were suitable for reconstructing the phylogeny of the cell
lineages, and whether phylogenetic trees adequately rep-
resented them, the robustness of the different nodes has
been measured, and independently estimated from two
different approaches. Bootstrap [20] was conducted with
1000 replicates of character resampling, and the highest
bootstrap percentages (BP) defined the strongest nodes.
The Bremer approach [21] measured the number of extra-
mutation-events required to break the corresponding
nodes, and the highest Bremer support indices (BSI) de-
fined the most robust nodes.
Preparation and hybridization of cDNA arrays
Variations in gene expression levels were analyzed by
large-scale measurement with home-made cDNA mini-ar-
rays (7.5 × 9 cm; 720 human genes; 11 genes/cm
2
) pro-
duced in our facility (TAGC, University of Marseille
Luminy). The spotted targets were PCR products ampli-
fied from control clones and IMAGE cDNA clones
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(IMAGE consortium, Hinxton, UK). Selected cDNA
clones corresponded to identified genes positioned on
chromosomes 1q and 17q. Information was gathered and
crosschecked from different web based data bases such as
genemap http://www.ncbi.nlm.nih.gov/genemap99/
,
Genecards http://genecards.weizmann.ac.il/
, Genelynx
http://www.genelynx.org/
or UCSC Genome http://ge-
nome.ucsc.edu/. PCR amplification and automatic spot-
ting of PCR products to the arrays (nylon Hybond-N+
membranes, Amersham Pharmacia Biotech; Little Chal-
font, UK) were performed according to Bertucci and col-
leagues (1999). Each array was hybridized with a
33
P-
labeled probe synthesized by reverse transcribing 5 µg of
total RNA for each sample [22]. Labeling of complex
probes, hybridization and washing conditions were as de-
scribed http://tagc.univ-mrs.fr/pub/cancer
. Arrays were
exposed to phosphor-imaging plates and then scanned
with a FUJI BAS 5000 beta imager (Raytest, Asnieres,
France). Hybridization signals were quantified with the
HDG Analyzer software (Genomic Solution, Ann Arbor,
MI, USA), by integrating all spot pixel intensities and re-
moving a spot background value determined in the neigh-
boring area.
Clustering analysis of gene expression data
Data display and analysis was performed using Excel soft-
ware (Microsoft, Richmond, WA, USA). Intensity values
were adjusted by a normalization step based on the DNA
quantification of each spot and the sum of intensities de-
tected in each experiment. Expression profiles were ana-
lyzed by hierarchical clustering using the Cluster program
developed by Eisen and colleagues [23] and represented
as a cladogram using the treeview software.
Results
MCF-7 variants
Originally we collected 9 MCF-7 sublines, MCF-7-ATCC,
MCF-7-R, MCF-7-O, MCF-7-MF and MCF-7-MG, MCF-7-
MVLN, MCF-7-MVLN-6ms7, MCF-7-MVLN-6ms8, as well
as a doxorubicin resistant cell line which was believed to
be a MCF-7 variant. All these sublines except the three
MCF-7-MVLN were of different origins with variable
number of passages and culture conditions.
MCF-7-MVLN, MCF-7-MVLN-6ms7 and MCF-7-MVLN-
6ms8 resulted from a selection process. MCF-7-MVLN
were transfected with an ERE-Luciferase construct [24]
and selected for Gentamycin resistance, while both MCF-
7-MVLN-6ms7 and MCF-7-MVLN-6ms8 have been pro-
duced by a long term exposure of MCF-7-MVLN cells to
200 nM OH-TAM. We also isolated cell clones from MCF-
7-R using limit dilution. Three clones MCF-7-R-F3, MCF-
7-R-D4 and MCF-7-R-G1 were selected for further studies.
This allowed us to verify that MCF-7 cells showed intrap-
opulational heterogeneity.
Common genetic origin of MCF-7 variants
Available information on the history of the different sub-
lines was not sufficient to rebuild lineages. It was, there-
fore, important to ascertain that all the tested sublines
bore a common genetic origin. To this end allelotypes at
9 polymorphic microsatellite markers located on 3 chro-
mosomal arms were determined. Eight of the 9 sublines
had identical haplotypes while the doxorubicin resistant
variant presented divergent allelic profiles at all markers
analyzed. This was therefore taken out of the study (data
not shown).
CGH analysis
Patterns of gains and losses shown by the different MCF-
7 variants were highly diverse (Table 1 and Figure 1).
Number of events ranged from 28 (MCF-7-ATCC) to 41
(MCF-7-MG) and, on average, losses were more frequent
(21) than gains (15). Only 9 events (6 losses, 3 gains)
were shared by the 11 cell lines (Figure 1). This small
number of common events could in part be attributed to
MCF-7-ATCC which presented the most divergent CGH
pattern. Out of the 28 gains or losses this subline dis-
played, 11 (6 losses, 5 gains) were specific to MCF-7-
ATCC cells. It was noticeable that the sizes of regions of
losses or gains varied according to the subline. This was
particularly striking for losses on 16q or gains at 3q or 5q
(Figure 1). Generally regions of gains tended to be more
heterogeneous in size and occurrence than losses.
These data were strong indications of the elevated level of
genetic heterogeneity shown by MCF-7 cells. It was, there-
fore, interesting to verify how cell lines of known filiation
compared to each other. Among all the cell lines tested
two such subsets were available to us, MCF-7-MVLN and
its two Tamoxifene resistant offshoots MCF-7-MVLN-
6ms7 and MCF-7-MVLN-6ms8, as well as MCF-7-R and
the three subclones we had derived; MCF-7-R-G1, MCF-7-
R-D4 and MCF-7-R-F3. MCF-7-MVLN cells and its variants
MCF-7-6ms7 and MCF-7-6ms8 presented rather homoge-
neous CGH patterns. Most anomalies found in MVLN
cells were also present in 6ms7 and 6ms8 to the exception
of 8 events; 1 gain (3p14) and 1 loss (6p22-p23) only
present in the Tamoxifene sensitive cells, 4 losses (3p21,
9q21-qter, 12q24-qter, 16pter-q11) and 2 gains (2p13-
p14, 4q22-q28) only present in the Tamoxifene resistant
variants. MCF-7-R and its descendants MCF-7-R-D4, G1
and F3 showed more heterogeneous profiles of gains and
losses. It was apparent that the 3 subclones derived from
MCF-7-R presented a larger number of events than the
mother line (Table 1). Seven regions of losses (4p16,
4q33-qter, 6p22-pter, 6q24-qter, 11p, 16p, 20p) and 6 re-
gions of gains (1q22-q25, 3p11-q23, 4q28-q32, 5q, 8q12-
q13, 12q13), observed in at least one of the subclones,
were absent in parent cells (Figure 2). Conversely, some
events present in parent cells were absent in at least one
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subclone (gains at 2q22-q33, 3p22-pter, 5p15, 7q21,
13q12-q14, 15q22-q25 or losses at 10q, 12q23-q24 and
16q11-q13). These data suggested that MCF-7-R bear a
higher level of clonal heterogeneity than MCF-7-MVLN
cells. Further indications of clonal heterogeneity in MCF-
7 cells could be found in the MCF-7-MF variant in which
the loss of chromosome 19, shared by all the variants, was
incomplete (i.e. the fluorescence ratio tumor DNA/nor-
mal DNA ranged between 1.0 and 0.75, whereas it was be-
low 0.75 in the other variants) (Figure 1).
Phylogenetic analysis
The diversity of CGH patterns illustrates the genomic plas-
ticity of MCF-7 cells and their capacity to acquire copy
number aberrations. It was thus interesting to verify
whether it was possible to reconstruct the phylogeny of
the MCF-7 variants studied. This question is directly relat-
ed to those classically addressed in evolutionary biology,
where different species are ordered and hierarchized ac-
cording to morphological and/or molecular characters.
Hence, computational methods developed in systematics
represented interesting tools to address the problem. We
chose to apply a character based approach called cladistics
under the maximum parsimony (MP) criterion, in which
Figure 1
CGH profiles of 11 MCF-7 sublines. Copy number alterations are indicated as bars on each side of the chromosome ideo-
grams, losses are shown by bars on the left, gains on the right. Each bar corresponds to an event observed in one subline.
Events indicated by dotted lines corresponded to gains or losses reproducibly observed but whose fluorescence ratios did not
reach the significance thresholds (1.3 or 0.75). Bars have been ordered from left to right for gains and from right to left for
losses. The relative order was (1) MCF-7-R, (2) MCF-7-R-D4, (3) MCF-7-R-G1, (4) MCF-7-R-F3, (5) MCF-7-MVLN, (6) MCF-7-
6ms7, (7) MCF-7-6ms8, (8) MCF-7-MF, (9) MCF-7-O, (10) MCF-7-MG, (11) MCF-7-ATCC.
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Table 1: Number of copy number alterations found in MCF-7 variants.
Variants Gains Losses Total number of events
MCF-7-R 18 16 34
MCF-7-R-D4 17 22 39
MCF-7-R-G1 18 20 38
MCF-7-R-F3 17 22 39
MCF7-MVLN-sens 15 22 37
MCF7-MVLN-6ms7 16 24 40
MCF7-MVLN-6ms8 15 21 36
MCF-7-MF 12 23 35
MCF-7-O 14 20 34
MCF-7-MG 18 23 41
MCF-7-ATCC 11 17 28
Figure 2
CGH profiles of MCF-7-R cells and its three subclones. Events were ordered from left to right for gains and from right to left
for losses. The relative order was (1) MCF-7-R, (2) MCF-7-R-D4, (3) MCF-7-R-G1, (4) MCF-7-R-F3. Circled events were spe-
cific to daughter clones. Boxed events correspond to gains or losses found only in the mother line (bold line) or in the mother
and one or two subclones (dotted boxes).
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different sublines were considered as taxa and copy
number changes at every chromosomal band as charac-
ters. We favored maximum parsimony because it allows to
order the different taxa and construct a phylogenetic tree
requiring the fewest number of changes. In such a tree
each cell line (or biological object) is represented as a leaf
while nodes correspond to a collection of inferred charac-
ters encountered in hypothetical ancestors. A supplemen-
tary advantage of maximum parsimony is that it allows
the identification of diagnostic events characterizing
groups of cell lines. Each chromosomal band was consid-
ered to exist under three discrete states; normal, loss, gain.
In the model we applied here, transformations from one
character to another were equally weighted. Although this
model did not perfectly match with CGH observations we
adopted it as the most workable approximation. As a mat-
ter of fact, cytogenetic bands vary greatly in size and a
number of them are below the resolution limit of CGH.
However, it is the only existing subdivision of chromo-
somal arms and it was not possible to base our analysis
using chromosomal arms as a unit because of an insuffi-
cient number of characters.
The maximum parsimony analysis was done twice. In the
first analysis we included only the 11 MCF-7 sublines and
defined a normal genome as the origin or root of our pu-
tative tree. In the second we included the doxorubicin re-
sistant cell line. Given its different genetic origin, it was
interesting to check how this cell line positioned relative
to bona fide MCF-7 variants in the phylogenetic tree. Fur-
thermore, the order of MCF-7-R and its subclones MCF-7-
R-G1, D4 and F3, as well as of MCF-7-MVLN and its
Tamoxifene resistant offshoots MCF-7-6ms7 and 6ms8
were important indications of the reliability of the phylo-
genetic reconstruction method used. Figure 3 shows the
phylogenetic tree corresponding to the analysis including
the 11 MCF-7 variants and the doxorubicin resistant line.
In this tree (or cladogram) doxorubicin resistant cells were
positioned as an external group and MCF-7-ATCC occu-
pied the position closest to the root, being the closest to a
common ancestor. The next clade was formed by MCF-7-
R which was identified as the ancestor of all other variants.
The remaining sublines were ordered as three broad
groups; one formed by MCF-7-MG, the second by the
three MCF-7-R subclones which formed a discrete clade
and the third with MCF-7-O, MCF-7-MF, MCF-7-MVLN
and both Tamoxifene resistant clones. We noted that
MCF-7-MVLN-6ms7 and MVLN formed a subgroup with-
in this clade, while MVLN-6ms8 were ordered at the same
level as MCF-7-MF or MCF-7-O.
Diagnostic characters
Diagnostic characters were identified using the analysis in
which only certified MCF-7 sublines had been included.
Characters were considered as diagnostic for a given clade
on the corresponding cladogram when they occurred once
and only once during the evolution of the 11 MCF-7 vari-
ants. Events (losses or gains) selected as diagnostic charac-
ters corresponded to minimal consensus regions.
Numbers of characters gradually added up when going
down the tree. As shown in Table 2, 8 events (5 losses and
3 gains) were identified as diagnostic characters of the
MCF-7 clade, since they were present in all the sublines,
including MCF-7-ATCC. The number of diagnostic char-
acters rose to 20 (9 corresponding to MCF-7-ATCC and 11
specific to R and its descendents) when MCF-7-R was tak-
en as a starting point, 22 with MCF-7-MG and 25 with
MCF-7-MVLN which is an endpoint on this tree.
Figure 3
Phylogenetic tree describing the relationships between the
MCF-7 sublines. The root was arbitrarily defined as corre-
sponding to a genome devoid of any CNA (normal genome).
The doxorubicin resistant line was also included in the analy-
sis. Since it did not belong to the MCF-7 group it qualified as
a potential outgroup and was indeed positioned as such by
the analysis. This tree is a consensus tree corresponding to
the 3 most parsimonious trees identified. It is 711 mutations
long. Values represented at the nodes correspond to boot-
strap percentages (top) and Bremer support indices (bot-
tom). These values measure the robustness of the nodes.
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RNA expression profiles
Because of the extent of the genomic changes shown by
different MCF-7 sublines it was important to assess the
consequences at the RNA expression level. We analyzed
RNA expression profiles of 8 MCF-7 sublines (MCF-7-
ATCC, MCF-7-O MCF-7-MF, MCF-7-MG, MCF-7-R, MCF-
7-R-G1, MCF-7-R-F3 and MCF-7-R-D4) along with those
of 19 unrelated breast cancer cell lines using home made
cDNA arrays comprising 721 genes localized on chromo-
some 1q and 17q respectively. Expression data were ana-
lyzed by hierarchical clustering using the Cluster program.
Seven of eight MCF-7 sublines were grouped within a clus-
ter gathering 10 cell lines, whereas MCF-7-ATCC was
grouped in an unrelated cluster (Figure 4A). Within the
MCF-7 cluster it was noticeable that 6 sublines (MF, O,
MG, R-F3, R-D4, R-G1) formed a tightly grouped subclus-
ter together with KPL-1 cells, while MCF-7-R was ordered
at a level equivalent to that of EFM-19 and EFM-192A
cells. The position of MCF-7-ATCC, which coclustered
with HCC 2218 and CAMA-1, was surprising. Although
some differences were foreseen these results went beyond
expectations and indicated the important distance this
subline showed with other MCF-7 variants. A further
question arose with the position of KPL-1 cells, which
grouped tightly with the MCF-7 subcluster. This raised
some doubt on the true identity of this cell line. Upon ver-
ification of its haplotype KPL-1 turned out to be identical
to MCF-7 cells. Altogether, these data show the elevated
dispersion of MCF-7 expression profiles. However, be-
cause genomic differences observed between MCF-7 vari-
ants were not restricted to chromosomes 1q and 17q, we
performed a complementary analysis on a set of 1000
genes selected for their proven or putative implication in
cancer [25]. These genes were localized on all chromo-
somes. Seven MCF-7 variants (MCF-7-MVLN, MCF-7-
ATCC, MCF-7-MF, MCF-7-MG, MCF-7-R, MCF-7-R-F3
and MCF-7-R-D4) were analyzed together with BT-474
breast cancer cells. Data were analyzed by hierarchical
clustering and the dendogram showed that while BT-474
behaved as an external group MCF-7-ATCC did not co-
cluster with other MCF-7 variants (Figure 4B). Although
the relative order found in this analysis is not identical to
the one found in the first analysis, results were in accord
confirming the divergence of MCF-7-ATCC cells.
Discussion
It is generally believed that divergence in cancer cell lines
is the consequence of differences in culture conditions,
Table 2: Diagnostic characters identified in the main nodes of the MCF-7 phylogenetic tree. Characters specific of each node (whose
occurrence has been associated with the emergence of the corresponding branch) are presented in bold type sets. Events in italics
correspond to characters passed on from ancestors.
MCF-7-ATCC MCF-7-R MCF-7-MG MCF-7-MVLN
Loss 1p32-p36 Loss 1p31-p36 Loss 1p31-p36 Loss 1p31-p36
Gain 1p13 Gain 1p13 Gain 1p13
Gain 1q31 Gain 1q31 Gain 1q31
Loss 11q42-q44 Loss 11q42-q44 Loss 11q42-q44
Loss 2q36-q37 Loss 2q36-q37 Loss 2q36-q37 Loss 2q36-q37
Gain 3q26 Gain 3q26 Gain 3q26
Loss 5q33
Loss 6q25-q27 Loss 6q25-q27
Gain 7q22 Gain 7q22 Gain 7q22
Loss 8p11-p23 Loss 8p11-p23 Loss 8p11-p23 Loss 8p11-p23
Gain 8q22-q23 Gain 8q22-q23 Gain 8q22-q23 Gain 8q22-q23
Loss 11q23-q25 Loss 11q23-q25 Loss 11q23-q25
Loss 12p13 Loss 12p13 Loss 12p13
Gain 12q15-q21 Gain 12q15-q21 Gain 12q15-q21
Loss 13q31-q34 Loss 13q31-q34 Loss 13q31-q34
Gain 14q21-q23 Gain 14q21-q24 Gain 14q21-q24 Gain 14q21-q24
Gain 15q26
Gain 16p11
Loss 17p11-p13 Loss 17p11-p13 Loss 17p11-p13
Gain 17q22-q24 Gain 17q22-q24 Gain 17q22-q24 Gain 17q22-q24
Loss 18q12-q23 Loss 18q12-q23 Loss 18q12-q23 Loss 18q12-q23
Loss 19p13-q13 Loss 19p13-q13 Loss 19p13-q13 Loss 19p13-q13
Loss 20p11-p13 Loss 20p11-p13
Gain 20q12-q13 Gain 20q12-q13 Gain 20q12-q13
Loss 21p13-q22 Loss 21p13-q22 Loss 21p13-q22
Page 8
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Page 9 of 12
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which change the selective pressure and, thus, favor the se-
lection of new genomic anomalies. If this situation is ex-
tended on a large number of cell passages it will lead to
important differences between cellular stocks. The level of
divergence can be directly related to that of genetic insta-
bility and breast cancer cell lines seem particularly prone
to it. Evidence for this can be found in recent work by Dav-
idson and colleagues [12] and Kytola and colleagues [26],
Figure 4
Hierarchical clustering of RNA expression profiles. Panel A clustering analysis of expression profiles of 8 MCF-7 sublines along
with those of 19 breast cancer cell lines. Expression profiling was done using home made Nylon arrays comprising 721 cDNAs
corresponding to identified genes localized on either chromosome 1q or 17q. Panel B clustering analysis of profiles of 7 MCF-7
sublines and the BT-474 breast cancer cell line. Nylon arrays comprised 1034 genes selected on the basis of their involvement
in cancer. Clustering analysis wass done on raw quantification results, which were just subjected to a scaling step but not to
ratio calculation. Parameters used in the analysis were Hierarchically Cluster Axes for Genesand Array: clusterand similarity
metric correlation centered with average linkage clustering. The dendogram on top of the diagram represents cell lines
ordered according to their degree of similarity. Complete datasets can be found at http://www.montp.inserm.fr/EMI0229/
download
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Page 10 of 12
(page number not for citation purposes)
who studied breast cancer cell lines using 24 color caryo-
typing or SKY. Seven cell lines were studied by both
groups and, for 3/7, reported data presented extensive dif-
ferences. Interestingly, MCF-7 cells were the most diver-
gent in both studies adding further evidence to existing
data on phenotypic or caryotypic variations in this cell
line. MCF-7 cells of different origins are characterized by
their variable chromosome numbers, which range from
55 to 90. Noticeably, some subsets present a bimodal
distribution with a first peak at 70 chromosomes and a
second one at 130 [8], indicating the coexistence of two
cellular subpopulations, one of which had undergone
endoreduplication.
Data presented here document that different MCF-7 vari-
ants underwent divergence at both the genomic and the
RNA expression levels. Furthermore, they indicate that
this can occur rapidly according to the MCF-7 variant con-
sidered. All the MCF-7 variants studied here showed
extensive differences in their CGH profiles. These differ-
ences affected the number of regions of either losses or
gains, which ranged from 28 in MCF-7-ATCC to 41 in
MCF-7-MG, as well as the size of the regions involved. Re-
markably, closely related sublines such as MCF-7-R and its
3 daughter clones MCF-7-R-D4, MCF-7-R-F3 and MCF-7-
R-G1 presented variations in their CGH profiles as well.
Daughter cells presented aberrations which were absent in
the mother subline and, this was less expected, had lost
anomalies present in the mother line. Furthermore, sister
clones showed different sets of anomalies indicating that
these cells bore the capacity to diverge over a limited
number of cell generations, even kept in identical culture
conditions. It is questionable whether this rapid upsurge
of anomalies fits a linear progression model, where muta-
tions are supposed to occur sequentially and be retained
due to positive selection. We think more plausible that the
differences shown by the 3 subclones be related to the ol-
igoclonal nature of MCF-7-R parent cells. Anomalies
found in the subclones in fact preexisted in MCF-7-R cells
and were brought to light by cell cloning. In comparison
MCF-7-MVLN and its two tamoxifene resistant derivatives
MCF-7-MVLN-6ms7 and MCF-7-MVLN-6ms8 were less
divergent. MCF-7-MVLN correspond to MCF-7 cells stably
transfected with ERE-Luciferase construct and went
through a gentamycin selection process. This could have
lead to the loss of the preexisting genetic heterogeneity.
We propose that MCF-7 cells contain an undetermined
number of coexisting clones, out of which one (or several)
possess stem clone potential and are responsible for the
genetic oligoclonality.
The oligoclonal nature of MCF-7 cells can be related to ab-
errant or instable mitoses. Indeed, cells that have lost
proper mitotic controls are prone to unequilibrated sister
chromatid exchanges and tolerate the propagation of
damaged chromosomes. As such they rapidly become
aneuploid and tend to accumulate physical aberrations.
Such anomalies have been reported in cellular models in
which the anaphase checkpoint gene MAD2 was disabled
[27–29], as well as in human tumors [30]. Consequently
instable mitoses will lead to rapid caryotypic changes. We,
thus, verified the integrity of the M phase in MCF-7-R
cells. MCF-7 cells did not show proper G2-M arrest when
challenged with Nocodazole, a spindle inhibitor (data not
shown). Our data are concordant with recently reported
data by Yoon and coworkers [31] which showed that 7/9
breast cancer cell lines, among which MCF-7, presented
important chromosome number variations.
The capacity to generate oligoclonality could be a strong
selective advantage for cancer cells because it allows for
rapid changes and as such confers an elevated genetic
plasticity. Such tumor systems would evolve according to
a nodal scheme (possibly through bursts) rather than fol-
lowing a linear selection model. Arguments in favor of a
nodal evolution scheme stemmed from the phylogenetic
analysis we have performed to reconstruct the history of
MCF-7 sublines and identify diagnostic characters
(CNAs). Because a number of analogies exist between ev-
olution of species and that of tumor cells, classification
methods developed for systematics have become increas-
ingly employed to analyze genetic data in cancer. Ap-
proaches, based on hierarchical clustering or other
distance-based models, have been applied to classify LOH
[32] or CGH results [33,34]. We chose the maximum par-
simony approach in a cladistic framework because it is a
character based classification method and, as such, was
considered to be best adapted to meet our goals [35]. We
reconstructed the phylogeny of the MCF-7 clade and, in-
terestingly, MCF-7-ATCC, which was the most divergent
MCF-7 subline in our study, was positioned closest to the
common ancestor. MCF-7-R came in second, positioned
as the ancestor of all other MCF-7 sublines. Out of the to-
tal of 62 CNAs present in all the sublines tested, only 8
were selected as diagnostic of the MCF-7 clade. This
means that this set of 8 events is shared by all the MCF-7
cells tested here and the original tumor possibly devel-
oped upon them. Thus, according to this phylogenetic
tree MCF-7-ATCC and MCF-7-R, which bear respectively
28 and 34 CNAs, evolved from a common node. The ro-
bustness of these results was reinforced by bootstrap and
Bremer analyses.
Given the extensive differences observed at the genomic
level we were interested to check different MCF-7 sublines
at the transcriptome level. Our RNA expression profiling
results confirmed the divergent position of MCF-7-ATCC
cells, which clustered with at some distance of other MCF-
7 sublines. It, thus, appears from the expression profiling
analysis, that MCF-7 sublines can show substantial
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Page 11 of 12
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differences at both the genomic and RNA expression lev-
els and this strongly suggests that the genomic differences
could translate into phenotypic differences of possibly
equivalent importance. MCF-7 cells are the most com-
monly used model for hormone responsive breast cancer
and there is generally little knowledge concerning the var-
iant used. Our data indicate that this may bear some im-
portance, given the level of genetic variability these cells
show and the rapidity with which they evolve.
Conclusions
In conclusion we want to propose that MCF-7 cells could
represent an interesting model for genetic evolution of a
subset of breast tumors. Breast tumors are prone to chro-
mosomal instability and frequently show cytogenetic oli-
goclonality [1]. While some cancers were shown to fit the
linear progression model, in which each step correspond-
ed to the occurrence of an additional anomaly [36], other
data brought evidence of more complex molecular
evolution schemes [37]. This latter study compared CGH
patterns of matched sets of primary breast tumors and
asynchronous metastases. A number of metastases fitted
the linear progression model, but it was noticeable that
some presented very divergent sets of anomalies com-
pared to their matched primary tumor. Only a limited set
of (in some cases none detectable) aberrations were
shared. The authors proposed the existence of a common
early stem clone which diverged, evolved independently
and ultimately lead to the formation of tumors with dif-
ferent locations. This scheme is very similar to what we
observed in MCF-7 cells when the ATCC subline was com-
pared to more distant offshoots of MCF-7-R. This leads us
to propose that the capacity to generate clonal heterogene-
ity could represents an important selective advantage in
some cancers and lead to aggressive and metastatic forms
of the disease.
Abbreviations
CNA, copy number alterations; CGH, comparative ge-
nomic hybridization; ERE, estrogen responsive element.
Competing interests
None declared.
Authors' contribution
MN carried out most of the cell culture work, CGH and ex-
pression array analyses. PC(1) set up and carried out the
analysis of CGH patterns by maximum parcimony as well
as the clustering analysis of RNA expression profiles. JV
helped setting up the expression profiling studies. BO co-
ordinated the CGH analysis of breast cancer cell lines. LU
carried out part of the CGH. CN helped setting up the ex-
pression profiling studies. DB helped setting up the ex-
pression profiling studies. EJPD helped carrying out the
maximum parcimony study. PC(2) helped setting up the
expression profiling technology in the lab. CT conceived
the study, its design and coordination and drafted the
manuscript.
Acknowledgments
This work was supported by funds from the Association pour la Recherche
sur le Cancer grant#5122, la Ligue Contre le Cancer program CIT, le Min-
istère de la Recherche et de la Technologie, Groupement des Entreprises
en Lutte contre le Cancer and Génopole Montpellier-Languedoc-Roussil-
lon. cDNA clones used to set up the arrays were provided by the HGMP
(UK-MRC, Cambridge, UK). The contribution of EJPD was the one 2003-
026 of the Institut des Sciences de l'Evolution de Montepellier (UMR 5554
CNRS). The authors wish to thank Drs Stephen Ethier, Alain Puisieux,
Françoise Vignon and Jean-Claude Nicolas for providing cell lines.
References
1. Heim S, Teixeira MR, Dietrich CU and Pandis N Cytogenetic poly-
clonality in tumors of the breast Cancer Genet.Cytogenet. 1997,
95:16-19
2. Flagiello D, Gerbault-Seureau M, Sastre-Garau X, Padoy E, Vielh P and
Dutrillaux B Highly recurrent der(1;16)(q10;p10) and other
16q arm alterations in lobular breast cancer [In Process
Citation] Genes Chromosomes Cancer 1998, 23:300-306
3. Teixeira MR, Pandis N, Bardi G, Andersen JA, Mitelman F and Heim S
Clonal heterogeneity in breast cancer: karyotypic compari-
sons of multiple intra- and extra-tumorous samples from 3
patients Int.J.Cancer 1995, 63:63-68
4. Visscher D, Dolan P, Ottosen S and Crissman J Geographic distri-
bution of co-dominant DNA stemlines in breast carcinoma
Cytometry 1995, 21:14-17
5. Bonsing BA, Corver WE, Fleuren GJ, Cleton-Jansen AM, Devilee P
and Cornelisse CJ Allelotype analysis of flow-sorted breast can-
cer cells demonstrates genetically related diploid and aneu-
ploid subpopulations in primary tumors and lymph node
metastases Genes Chromosomes Cancer 2000, 28:173-183
6. Soule HD, Vazguez J, Long A, Albert S and Brennan M A human cell
line from a pleural effusion derived from a breast carcinoma
J Natl Cancer Inst 1973, 51:1409-1416
7. Seibert K, Shafie SM, Triche TJ, Whang-Peng JJ, O'Brien SJ, Toney JH,
Huff KK and Lippman ME Clonal variation of MCF-7 breast can-
cer cells in vitro and in athymic nude mice Cancer Res 1983,
43:2223-2239
8. Whang-Peng J, Lee EC, Kao-Shan CS, Seibert K and Lippman M Cy-
togenetic studies of human breast cancer lines: MCF-7 and
derived variant sublines J Natl Cancer Inst 1983, 71:687-695
9. Butler WB, Berlinski PJ, Hillman RM, Kelsey WH and Toenniges MM
Relation of in vitro properties to tumorigenicity for a series
of sublines of the human breast cancer cell line MCF-7 Cancer
Res 1986, 46:6339-6348
10. Osborne CK, Hobbs K and Trent JM Biological differences
among MCF-7 human breast cancer cell lines from different
laboratories Breast Cancer Res Treat 1987, 9:111-121
11. Resnicoff M, Medrano EE, Podhajcer OL, Bravo AI, Bover L and Mor-
doh J Subpopulations of MCF7 cells separated by Percoll gra-
dient centrifugation: a model to analyze the heterogeneity of
human breast cancer Proc Natl Acad Sci U S A 1987, 84:7295-7299
12. Davidson JM, Gorringe KL, Chin SF, Orsetti B, Besret C, Courtay-Ca-
hen C, Roberts I, Theillet C, Caldas C and Edwards PA Molecular
cytogenetic analysis of breast cancer cell lines Br J Cancer 2000,
83:1309-1317
13. Jones C, Payne J, Wells D, Delhanty JD, Lakhani SR and Kortenkamp
A Comparative genomic hybridization reveals extensive var-
iation among different MCF-7 cell stocks Cancer Genet Cytogenet
2000, 117:153-158
14. Courjal F, Cuny M, Rodriguez C, Louason G, Speiser P, Katsaros D,
Tanner MM, Zeillinger R and Theillet C DNA amplifications at
20q13 and MDM2 define distinct subsets of evolved breast
and ovarian tumours Br.J.Cancer 1996, 74:1984-1989
15. Orsetti B, Courjal F, Cuny M, Rodriguez C and Theillet C 17q21-q25
aberrations in breast cancer: combined allelotyping and
CGH analysis reveals 5 regions of allelic imbalance among
Page 11
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BMC Cancer 2003, 3 http://www.biomedcentral.com/1471-2407/3/13
Page 12 of 12
(page number not for citation purposes)
which two correspond to DNA amplification [In Process
Citation] Oncogene 1999, 18:6262-6270
16. Courjal F and Theillet C Comparative genomic hybridization
analysis of breast tumors with predetermined profiles of
DNA amplification Cancer Res. 1997, 57:4368-4377
17. Fitch WM Toward defining the course of evolution: minimum
change for a specific tree topology Syst. Zool. 1971, 20:406-416
18. Swofford DL PAUP*. Phylogenetic Analysis Using Parsimony
(* and Other Methods). Version 4 Sunderland, Massachusetts, Sin-
auer Associates 1998,
19. Maddison WP and Maddison DR MacClade: analysis of phylogeny
and character evolution, version 3.04 Sunderland, MA, Sinauer
Associates 1992,
20. Felsenstein J Confidence limits on phylogenies: an approach
using the bootstrap Evolution 1985, 39:783-791
21. Bremer K Branch support and tree stability Cladistics 1994,
10:295-304
22. Bertucci F, Bernard K, Loriod B, Chang YC, Granjeaud S, Birnbaum
D, Nguyen C, Peck K and Jordan BR Sensitivity issues in DNA ar-
ray-based expression measurements and performance of ny-
lon microarrays for small samples Hum Mol Genet 1999, 8:1715-
1722
23. Eisen MB, Spellman PT, Brown PO and Botstein D Cluster analysis
and display of genome-wide expression patterns Proc Natl Acad
Sci U S A 1998, 95:14863-14868
24. Pons M, Gagne D, Nicolas JC and Mehtali M A new cellular model
of response to estrogens: a bioluminescent test to character-
ize (anti) estrogen molecules Biotechniques 1990, 9:450-459
25. Bertucci F, Viens P, Hingamp P, Nasser V, Houlgatte R and Birnbaum
D Breast cancer revisited using DNA array-based gene ex-
pression profiling Int J Cancer 2003, 103:565-571
26. Kytola S, Rummukainen J, Nordgren A, Karhu R, Farnebo F, Isola J and
Larsson C Chromosomal alterations in 15 breast cancer cell
lines by comparative genomic hybridization and spectral
karyotyping Genes Chromosomes Cancer 2000, 28:308-317
27. Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald
W, Dobles M, Sorger PK, Murty VV and Benezra R MAD2 haplo-
insufficiency causes premature anaphase and chromosome
instability in mammalian cells Nature 2001, 409:355-359
28. Jallepalli PV, Waizenegger IC, Bunz F, Langer S, Speicher MR, Peters
JM, Kinzler KW, Vogelstein B and Lengauer C Securin is required
for chromosomal stability in human cells Cell 2001, 105:445-
457
29. Wang X, Jin DY, Ng RW, Feng H, Wong YC, Cheung AL and Tsao
SW Significance of MAD2 expression to mitotic checkpoint
control in ovarian cancer cells Cancer Res 2002, 62:1662-1668
30. Saunders WS, Shuster M, Huang X, Gharaibeh B, Enyenihi AH, Pe-
tersen I and Gollin SM Chromosomal instability and cytoskele-
tal defects in oral cancer cells Proc Natl Acad Sci U S A 2000,
97:303-308
31. Yoon DS, Wersto RP, Zhou W, Chrest FJ, Garrett ES, Kwon TK and
Gabrielson E Variable levels of chromosomal instability and
mitotic spindle checkpoint defects in breast cancer Am J Pathol
2002, 161:391-397
32. van Tilborg AA, de Vries A, de Bont M, Groenfeld LE, van der Kwast
TH and Zwarthoff EC Molecular evolution of multiple recur-
rent cancers of the bladder Hum Mol Genet 2000, 9:2973-2980
33. Jiang F, Desper R, Papadimitriou CH, Schaffer AA, Kallioniemi OP,
Richter J, Schraml P, Sauter G, Mihatsch MJ and Moch H Construc-
tion of evolutionary tree models for renal cell carcinoma
from comparative genomic hybridization data Cancer Res
2000, 60:6503-6509
34. Kainu T, Juo SH, Desper R, Schaffer AA, Gillanders E, Rozenblum E,
Freas-Lutz D, Weaver D, Stephan D, Bailey-Wilson J, Kallioniemi OP,
Tirkkonen M, Syrjakoski K, Kuukasjarvi T, Koivisto P, Karhu R, Holli
K, Arason A, Johannesdottir G, Bergthorsson JT, Johannsdottir H,
Egilsson V, Barkardottir RB, Johannsson O, Haraldsson K, Sandberg
T, Holmberg E, Gronberg H, Olsson H, Borg A, Vehmanen P, Eerola
H, Heikkila P, Pyrhonen S and Nevanlinna H Somatic deletions in
hereditary breast cancers implicate 13q21 as a putative nov-
el breast cancer susceptibility locus Proc Natl Acad Sci U S A 2000,
97:9603-9608
35. Planet PJ, DeSalle R, Siddall M, Bael T, Sarkar IN and Stanley SE Sys-
tematic analysis of DNA microarray data: ordering and in-
terpreting patterns of gene expression Genome Res 2001,
11:1149-1155
36. Larramendy ML, Lushnikova T, Bj]orkqvist AM, Wistuba ,II, Virmani
AK, Shivapurkar N, Gazdar AF and Knuutila S Comparative ge-
nomic hybridization reveals complex genetic changes in pri-
mary breast cancer tumors and their cell lines Cancer Genet
Cytogenet 2000, 119:132-138
37. Kuukasjarvi T, Karhu R, Tanner M, Kahkonen M, Schaffer A, Nuppo-
nen N, Pennanen S, Kallioniemi A, Kallioniemi OP and Isola J Genetic
heterogeneity and clonal evolution underlying development
of asynchronous metastasis in human breast cancer Cancer
Res. 1997, 57:1597-1604
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  • Source
    • "Whilst they serve as useful tools, there are significant limitations, because continual passage of these cell lines is accompanied by extensive clonal selection and consequent loss of heterogeneity [19,20]. Moreover, different isolates of the same cell line can differ from one another at both the genomic and gene expression levels [21]. Their lack of predictive value is highlighted by the absence of correlation between clinical results and in vitro and in vivo data obtained with cell lines [22], in part contributing to the >90% failure rate for the development of new oncology drugs [1] . "
    [Show abstract] [Hide abstract] ABSTRACT: Despite advances in the treatment of patients with early and metastatic breast cancer, mortality remains high due to intrinsic or acquired resistance to therapy. Increased understanding of the genomic landscape through massively parallel sequencing has revealed somatic mutations common to specific subtypes of breast cancer, provided new prognostic and predictive markers, and highlighted potential therapeutic targets. Evaluating new targets using established cell lines is limited by the inexact correlation between responsiveness observed in cell lines versus that elicited in the patient. Patient-derived xenografts (PDXs) generated from fresh tumor specimens recapitulate the diversity of breast cancer and reflect histopathology, tumor behavior, and the metastatic properties of the original tumor. The high degree of genomic preservation evident across primary tumors and their matching PDXs over serial passaging validate them as important preclinical tools. Indeed, there is accumulating evidence that PDXs can recapitulate treatment responses of the parental tumor. The finding that tumor engraftment is an independent and poor prognostic indicator of patient outcome represents the first step towards personalized medicine. Here we review the utility of breast cancer PDX models to study the clonal evolution of tumors and to evaluate novel therapies and drug resistance.
    Full-text · Article · Dec 2015 · Breast Cancer Research
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    • "On the other hand, Ao et al. observed that tamoxifen attenuated mammosphere formation from MCF7 cells [48]. Importantly, all studies have been performed with cancer cell lines that have accumulated genetic modifications after decades of in vitro culturing [49] . Hence, it is important that clinically relevant conclusions are generated from patient-derived cells without ability to adapt to an in vitro environment. "
    [Show abstract] [Hide abstract] ABSTRACT: Breast cancer cells with stem cell characteristics (CSC) are a distinct cell population with phenotypic similarities to mammary stem cells. CSCs are important drivers of tumorigenesis and the metastatic process. Tamoxifen is the most widely used hormonal therapy for estrogen receptor (ER) positive cancers. In our study, tamoxifen was effective in reducing proliferation of ER + adherent cancer cells, but not their CSC population. We isolated, expanded and incubated CSC from seven breast cancers with or without tamoxifen. By genome-wide transcriptional analysis we identified tamoxifen-induced transcriptional pathways associated with ribosomal biogenesis and mRNA translation, both regulated by the mTOR-pathway. We observed induction of the key mTOR downstream targets S6K1, S6RP and 4E-BP1 in-patient derived CSCs by tamoxifen on protein level. Using the mTOR inhibitors rapamycin, everolimus and PF-04691502 (a dual PI3K/mTOR inhibitor) and in combination with tamoxifen, significant reduction in mammosphere formation was observed. Hence, we suggest that the CSC population play a significant role during endocrine resistance through activity of the mTOR pathway. In addition, tamoxifen further stimulates the mTOR-pathway but can be antagonized using mTOR-inhibitors. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Jul 2015 · Cancer letters
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    • "Changes in the expressions of repair proteins BRCA-1/BRCA-2 and wild type p53 are regarded as the contributors for MDR develop- ment [40]. The mechanisms of drug resistance development may be operated within proliferating MCF-7 populations to generate phenotypic diversity continuously [41] . Thus, genetic heterogeneity could be suggested among the series of MCF-7/ADR-n cell lines. "
    [Show abstract] [Hide abstract] ABSTRACT: Cellular mechanisms of multidrug resistance (MDR) are related to ABC transporters, apoptosis, antioxidation, drug metabolism, DNA repair and cell proliferation. It remains unclear whether the process of resistance development is programmable. We aimed to study gene expression profiling circumstances in MCF-7 during MDR development. Eleven MCF-7 sublines with incremental doxorubicin resistance were established as a valued tool to study resistance progression. MDR marker P-gp was overexpressed only in cells termed MCF-7/ADR-1024 under the selection dose approaching 1024 nM. MCF-7/ADR-1024 and authentic MCF-7/ADR shared common features in cell morphology and DNA ploidy status. MCF-7/ADR-1024 and authentic MCF-7/ADR down regulated repair genes BRCA1/2 and wild type p53, apoptosis-related gene Bcl-2 and epithelial-mesenchymal transition (EMT) epithelial marker gene E-cadherin. While detoxifying enzymes glutathione-S transferase-π and protein kinase C-α were up-regulated. The genes involving in EMT mesenchymal formation were also overexpressed, including N-cadherin, vimentin and the E-cadherin transcription reppressors Slug, Twist and ZEB1/2. PI3K/AKT inhibitor wortmannin suppressed expression of Slug, Twist and mdr1. Mutant p53 with a deletion at codons 127-133 markedly appeared in MCF-7/ADR-1024 and authentic MCF-7/ADR as well. In addition, MCF-7/ADR-1024 cells exerted CSC-like cell surface marker CD44 high/CD24 low and form mammospheres. Overall, results suggest that resistance marker P-gp arises owing to turn on/off or mutation of the genes involved in DNA repair, apoptosis, detoxifying enzymes, EMT and ABC transporters at a turning point (1.024 μM doxorubicin challenge). Behind this point, no obvious alterations were found in most tested genes. Selection for CSC-like cells under this dose may importantly attribute to propagation of the population presenting invasive properties and drug resistance. We thereby suggest two models in the induction of drug resistance. Model 1: Selection for CSC-like cells. Model 2: Mutations for gain-of resistance. Either model 1 or model 2 requires doxorubicin dose approaching 1 μM to alter gene regulation.
    Full-text · Article · Jan 2015 · PLoS ONE
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