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Cancer Genetics and Cytogenetics 163 (2005) 44–56
Chromosomal alterations cause the high rates and wide ranges
of drug resistance in cancer cells
Ruhong Lia, Ruediger Hehlmanb, Rainer Sachsc, Peter Duesberga,*
b
a
Department of Molecular and Cell Biology, Donner Laboratory, University of California, Berkeley, Berkeley, CA 94720
III. Medizinische Klinik Mannheim of the University of Heidelberg at Mannheim, Wiesbadener Str. 7-11, 68305 Mannheim, Germany
c
Departments of Mathematics and Physics, Evans Hall, University of California, Berkeley, Berkeley, CA 94720
Received 16 December 2004; received in revised form 30 April 2005; accepted 2 May 2005
Abstract
Conventional mutation-selection theories have failed to explain (i) how cancer cells become spontaneously resistant against cytotoxic drugs at rates of up to 1023 per cell generation, orders higher
than gene mutation, even in cancer cells; (ii) why resistance far exceeds a challenging drugda state
termed multidrug resistance; (iii) why resistance is associated with chromosomal alterations and
proportional to their numbers; and (iv) why resistance is totally dependent on aneuploidy. We propose here that cancer-specific aneuploidy generates drug resistance via chromosomal alterations.
According to this mechanism, aneuploidy varies the numbers and structures of chromosomes automatically, because it corrupts the many teams of proteins that segregate, synthesize, and repair chromosomes. Aneuploidy is thus a steady source of chromosomal variation from which, in classical
Darwinian terms, resistance-specific aneusomies are selected in the presence of chemotherapeutic
drugs. Some of the thousands of unselected genes that hitchhike with resistance-specific aneusomies can thus generate multidrug resistance. To test this hypothesis, we determined the rates of
chromosomal alterations in clonal cultures of human breast and colon cancer lines by dividing
the fraction of nonclonal karyotypes by the number of generations of the clone. These rates were
about 1022 per cell generation, orders higher than mutation. Chromosome numbers and structures
were determined in metaphases hybridized with color-coded chromosome-specific DNA probes.
Further, we tested puromycin-resistant subclones of these lines for resistance-specific aneusomies.
Resistant subclones differed from parental lines in four to seven specific aneusomies, of which different subclones shared some. The degree of resistance was roughly proportional to the number of
these aneusomies. Thus, aneuploidy is the primary cause of the high rates and wide ranges of drug
resistance in cancer cells. Ó 2005 Elsevier Inc. All rights reserved.
1. Introduction
Conventional mutation-selection theories have failed to
answer four critical sets of questions about drug resistance
in cancer cells.
First: How do cancer cells become spontaneously resistant against cytotoxic drugs at the exceedingly high rates
of up to 1023 per cell generation [1–6], whereas for mutation the rate per cell generation is restricted to 1027 for dominant and to 10214 for recessive genes [6–10], even in most
cancer cells [9–18]? How can an initial low resistance be
enhanced stepwise by more than 1,000-fold [1,3–5,19–28],
although multiple mutations of specific genes are extremely
* Corresponding author. Tel.: (510) 642-6549; fax: (510) 643-6455.
E-mail address: [email protected] (P. Duesberg).
0165-4608/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.cancergencyto.2005.05.003
unlikely in view of the low rates of conventional gene mutation? And how does resistance acquired by many, but
not all, cancer cells revert in the absence of selective drugs
at about the same high rates at which it was acquired
[3–5,23,26,27,29–32]? And why are many cancer cells even
intrinsically, or a priori, resistant to cytotoxic drugs, which
kill normal cells by definition [24,33,34]?
Second: How does the range of drug resistance of cancer
cells far exceed a challenging drug, including drugs that are
entirely unrelated to the one that induced it [5,28,35–41]?
(That state, termed multidrug resistance, is the nemesis of
chemotherapy [5,35].) Why is multidrug resistance so common, although simultaneous mutation of multiple genes is
extremely unlikely [5,28,33,35–40]? And why are many
cancers intrinsically multidrug resistant, although spontaneous mutation is very rare [24,34]? And how do cancer cells
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
from which putative multidrug resistance genes have been
deleted become multidrug resistant, as we have found
recently [4]?
Third: Why do drug-resistant cells differ from sensitive
precursor cells in chromosomal alterations [21–23,30,36,
42–62], including extra- and intrachromosomal amplicons
[26,27,31,63,64]? And why is resistance proportional to
the numbers of altered chromosomes or aneusomies
[28,36,37,49]?
Fourth and last: Why is the generation of drug resistance
totally dependent on aneuploidy [3,4,6]?
These idiosyncratic features of drug resistance in cancer
cells have been difficult to reconcile with conventional
genetic theories [2,24,65,66]. Therefore, either ‘‘not welldefined [gene] activation’’ [5] or ‘‘epigenetic’’ mechanisms
[6,24,50,65] or as yet unidentified mechanisms of variation
[2,32,50,67] have been postulated.
We propose here that cancer-specific aneuploidy generates drug resistance. According to this mechanism, aneuploidy varies the numbers and structures of chromosomes
automatically, because it corrupts the stoichiometry and
functions of the many teams of proteins that segregate, synthesize, and repair chromosomes [16,68,69]. The resulting
nondisjunctions change the numbers of chromosomes and
the resulting DNA breaks initiate chromosomal deletions,
rearrangements, and amplifications [16,64,69,70], including
intra- and extrachromosomal amplicons, or what are known
as homogeneously staining regions and double minute chromosomes [26,63,64]. Aneuploidy is thus a steady source of
chromosomal variation from which, in classical Darwinian
terms, selection in the presence of chemotherapeutic drugs
would encourage the emergence of new drug-resistant cell
species with resistance-specific aneusomies. The new or
altered transcriptomes generated by resistance-specific
chromosome alterations [50,71,72] would then produce
resistance-specific functions. Some of the thousands of
unselected genes that hitchhike with resistance-specific
aneusomies can thus generate multidrug resistance.
To test this model, we first asked whether the rates of
chromosomal variations in cancer cells are indeed higher
than those of conventional mutation. These rates were determined by measuring the fraction of karyotypes with nonclonal chromosomal variations in clonal cultures of three
human breast and colon cancer cell lines: MDA 231, SW
480, and HT 29. The rates of chromosomal variations per
cell generation were then calculated by dividing these fractions by the number of generations of the clonal culture.
Variations of the numbers and structures of chromosomes
were detected in metaphases hybridized in situ with fluorescent color-coded chromosome-specific DNA probes following published procedures [73,74]. Next we have asked,
whether puromycin-resistant subclones of these cell lines
differ from the sensitive parental lines in resistance-specific
aneusomies, using the same method to identify numerically
and structurally altered chromosomes. Puromycin, which is
an inhibitor of protein synthesis, was chosen as the cytotoxic
45
drug, because it is neither mutagenic nor aneuploidogenic
[75], and thus is unlikely to confound resistance-specific
karyotypic alterations by generating artificial chromosomal
alterations on its own. The drug has also been used as an
antineoplastic drug in the past [76].
2. Materials and methods
2.1. Cell lines
The human colon cancer cell lines HT 29 and SW 480
were obtained from the American Type Culture Collection
(ATCC, Rockville, MD). The human breast cancer line
MDA 231 was a kind gift of Edward Gabrielson (Johns
Hopkins University, Baltimore, MD) [77]. Cell lines were
propagated in RPMI 1640 medium supplemented with
3% fetal calf serum, 1% calf serum, and antibiotics (Gibco
BRL, Gaithersburg, MD; Sigma-Aldrich, St. Louis, MO).
Prior to the selection of puromycin-resistant and random
subclones, each human cancer cell line was cloned to eliminate nonclonal variants from prior cell generations (see
section 3.1) [68,69]. Clones were prepared either by plating
~100 cells on 10-cm culture dishes and selecting discrete
colonies or by seeding an average of 0.5 cell into each well
of a multi-well cloning dish and harvesting the resulting
colonies following published procedures [3,73].
2.2. Preparation of puromycin-resistant subclones
from cancer cell lines by the classic stepwise
selection protocol
Following a published classic stepwise selection protocol [5], selection of variants from the cancer cell lines with
resistance to puromycin (Sigma-Aldrich) was initiated at
low micromolar concentrations, which were increased stepwise in subsequent cultures to obtain more and more resistant variants [1,3,4,20]. For this purpose, ~5 106 cells of
the cancer cell lines studied were first incubated for between 1 and 2 weeks with 1–2 mg puromycin per 10-cm
dish containing 7–10 mL medium. Different subcultures
were then treated with either small (0.5–1 mg) drug increments, which were survived by most cells, or with bigger
increments, survived by only a small minority of cells. During this selection, at escalating concentrations of puromycin, confluent cultures of cells were transferred after 1:2
to 1:4 dilutions about two times per month, which corresponds to two to four cell generations. After an average
of 4 to 6 months or 8 to 24 cell generations, the following
seven puromycin-resistant subclones were obtained: HT 29
P1 (9), P2 (3), and P3 (3); SW 480 P1 (8) and P2 (6); MDA
231 P1 (5) and P2 (4). The numbers in parentheses indicate
the concentrations of puromycin (in mg per 10 cm-dish) at
which these cultures were analyzed, as described in section
3.2. By contrast, the drug-sensitive parental lines would die
within 4 to 6 days at such concentrations of puromycin.
46
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
2.3. Karyotyping by in situ hybridization of
metaphases with fluorescent color-coded,
chromosome-specific DNA probes
Metaphase chromosomes were prepared and hybridized
in situ with fluorescent color-coded, chromosome-specific
probes purchased from MetaSystems (Boston, MA). The
exact methods for multicolor fluorescence in situ hybridization are described by the manufacturer (MetaSystems) and
by us previously [73,74].
2.4. Rates of spontaneous chromosomal variations
of three human cancer lines
To determine the rates at which the three human cancer
cell lines HT 29, SW 480, and MDA 231 spontaneously
generate new variant karyotypes, two similar methods were
used. One method calculates these rates from the percentage of cells with new clonal chromosomal variations in
a clonal culture. The rate of chromosome alteration per cell
generation is then obtained by dividing this percentage by
the number of cell generations since the clone was initiated.
This method, which we call the karyotype method, is biased
in favor of the rates at which viable and competitive chromosomal alterations occur, except for potentially nonviable
or noncompetitive karyotypes from the last clonal generation prior to karyotyping. The other method, which we call
the subcloning method, calculates the rate of alteration from
the percentage of viable, new variants with new clonal
chromosomal variations among subclones derived from
a given clone. The rate of chromosome alteration per cell
generation is then also obtained by dividing this percentage
by the number of cell generations since the clone was
initiated.
3. Results
3.1. Spontaneous karyotype variation
3.1.1. HT 29
The karyotypes of 24 metaphases of a clonal culture of
the colon cancer cell line HT 29 are shown in Table 1. This
line has a modal chromosome number of 71 and is thus
highly aneuploid. In agreement with a previous study, this
cell line contains 38 distinct chromosomes, which include
15 clonal and 6 nonclonal marker chromosomes [78]. The
clonal hybrid or marker chromosomes were labeled M1–
M15, followed by the numbers of the original chromosomal
constituents in their relative sequences (e.g., M7 9/20/9
represents marker chromosome 7, with its hybrid origins
sequentially from chromosomes 9, 20, and again 9). The
nonclonal markers were labeled according to the same
scheme, with numbering picking up where the clonal
markers left off. A representative karyotype prepared with
color-coded chromosome-specific probes is shown in
Fig. 1A.
As seen from Table 1, 8 out of 24 (33%) cells of the HT
29 clone had identical karyotypes. By contrast, the karyotypes of 16 out of 24 (66%) cells of the clone differed from
the predominant clonal (and presumed to be the parental)
karyotype in one or more nonclonal (nonparental) numerical or structural chromosome alterations (highlighted in
Table 1). The predominant subclone of a cancer or of a
clonal culture of cancer cells is also called the stemline
[42,43,79,80]. Seven of the 16 nonparental variants of the
HT 29 clone differed from the stemline in a single alteration, and 9 of the 16 differed in multiple alterations. These
nine nonparental karyotypes were grouped into four subclones, because they shared several new numerical or structural chromosome alterations (highlighted in Table 1). One
of the alterations was an exact dimer (2n) of the stemline
and another was a near dimer (see metaphases 23 and 24,
Table 1). Similar polyploidizations of neoplastic cells have
been observed previously by others and by us [69,81–85]
(see also Table 3).
To determine the rate of chromosomal variation per cell
generation by the karyotype method, all cells with identical, nonparental karyotypes (i.e., subclones) were counted
as one variant, because they are the progeny of the same
variation of the stemline. Thus, the percentage of new nonparental karyotypes in our HT 29 clone was 46% (5 11:24,
or 4 nonparental subclones 1 7 singular nonparental cells
per 24 clonal HT 29 cells). Because the clone had gone
through about 23 cell generations by the time it was analyzed, and had reached about 107 cells, the rate of chromosomal alteration per HT 29 cell generation was 46% over
the 23 generations (46%/23), or 2%, according to the karyotype method.
A slightly lower rate of karyotype variation was obtained
by the subcloning method, which detects only viable and
competitive karyotypic variants. According to this method,
the rate at which the HT 29 line generates new viable karyotypes was 1.4% (33%/23) per generation, because only
one of three randomly selected subclones of HT 29 (i.e.,
HT 29 R3) differed from the parental stemline in one clonal
chromosomal variation (Table 1). The karyotypes of the
other two subclones, R1 and R2, were identical to the
parental karyotype, except for the expected random, nonclonal aneusomies in some metaphases, which reflect the
same general chromosomal instability as the parent line
(data not shown).
3.1.2. SW 480
The karyotypes of 19 cells of a clonal culture of the colon cancer cell line SW 480 are shown in Table 2. The SW
480 line resembled the HT 29 line as follows: SW 480 is
aneuploid, having a modal chromosome number of 57,
and contains 15 clonal marker chromosomes (numbered
M1–M15) and six nonclonal markers (numbered M16–
M21). A representative karyotype prepared with colorcoded chromosome-specific probes, is shown in Fig. 1C.
As seen from Table 2, 6 out of 19 (32%) SW 480 cells
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
47
Table 1
Karyotypes of a clonal culture of colon cancer line HT 29, of a distinct subclone R3, and of three puromycin-resistant subclones P1, P2 and P3
Metaphases
Clones
Chromosomes
1–8*
HT 29
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
M1 81
M2 3/?/3
M3 131
M4 6/14
M5 3/?
M6 D131
M7 9/20/9
M8 X2
M9 191
M10 17/22/17
M11 17/19
M12 D6/14
M13 62
M14 5/20
M15 182
M 16 13/5
M 17 2/17
M 18 17/1
M 19 9/20/9/6
M 20 2/13
DM chr. 5
19
4
6
7
13
M14 5/20
M16 13/5
6
11
21
M9 191
M17 11/19/21
9
11
21
M3 131
M14 5/20
M18 11/21
3
3
1
3
3
1
4
1
2
3
4
3
1
1
4
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
2
2
3
0
0
1
0
3
1
0
1
1
3
1
0
2
1
HT 29-R3
HT 29-P1
HT 29-P2
HT 29-P3
10
15
20
3
3
1
3
3
1
4
1
2
3
4
3
1
1
3
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
3
3
1
3
3
1
4
1
2
3
4
3
1
1
3
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
3
3
1
3
3
1
4
1
2
3
4
3
1
1
3
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
3
3
1
3
3
0
4
1
2
3
3
3
1
1
4
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
3
3
1
3
3
0
4
1
2
3
3
3
1
1
4
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
3
3
1
3
3
2
3
1
2
3
4
3
1
2
4
3
2
2
2
4
2
2
2
1
1
1
0
1
1
0
1
0
2
1
0
0
1
1
0
1
1
1
0
0
1
3
3
1
3
3
2
3
1
2
3
4
3
0
2
4
3
2
2
2
4
2
2
2
1
1
1
0
1
1
0
1
0
2
1
0
0
1
1
0
1
1
1
0
0
1
3
3
1
3
3
1
4
1
2
3
4
2
1
1
4
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
1
3
3
1
3
3
1
3
1
2
3
4
2
1
1
4
3
2
2
2
4
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
1
0
3
1
0
1
1
3
1
0
2
1
0
3
1
0
1
1
3
1
0
2
1
0
3
1
0
1
1
3
1
0
2
1
0
3
1
0
1
1
3
1
0
2
1
0
3
1
0
1
1
3
1
0
2
1
0
3
1
0
1
1
3
1
0
2
1
0
3
1
0
1
1
3
1
0
2
1
0
2
1
0
1
1
3
1
0
1
1
0
2
1
0
1
2
6
2
0
4
2
*, identical metaphases; bold, non-clonal aneusomies; DM, double minute.
3
3
1
3
3
1
4
1
2
3
4
3
1
1
4
2
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
3
4
1
3
3
1
4
1
2
3
4
3
1
1
4
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
3
3
1
3
3
1
5
1
2
3
4
3
1
1
4
3
2
2
2
4
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
24
3
3
1
3
2
1
4
1
2
3
4
3
1
1
4
3
2
1
2
4
2
2
2
1
1
1
1
1
1
1
1
1
0
1
1
1
2
1
0
0
0
0
0
0
2
3
1
3
3
1
4
1
2
3
4
3
0
1
4
3
2
2
2
3
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
0
0
0
0
0
6
6
2
6
6
2
8
2
4
6
8
6
2
2
8
6
4
4
4
8
4
4
4
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
7
4
2
5
9
2
10
2
5
6
7
6
2
0
8
6
3
4
5
10
4
5
7
2
2
1
3
4
2
2
2
2
1
2
2
2
2
2
0
0
0
0
1
9
48
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
had identical karyotypes, and 13 differed from the 6 in one
or more numerical or structural alterations (highlighted in
Table 2). Of the 13 variant karyotypes, 6 differed from
the stemline in a single alteration and 7 differed in multiple
alterations. Four of those seven variant karyotypes with
multiple alterations can be grouped into two subclones
sharing two identical numerical chromosome alterations:
namely, metaphases 7 and 8 and 9 and 10 in Table 2. Three
more metaphases may be subclones of the subclone 9 or 10
in Table 2, or may be independent, because they differ from
9 and 10 in further aneusomies (Table 2). Thus, there were
a total of 11 new subclonal and nonclonal variants in a total
of 19 clonal SW 480 cells.
According to the karyotype method, the rate of chromosomal variation per cell generation was 2.5% that is, 58%
(5 11:19, or 2 new subclonal 1 9 singular nonclonal cells
per 19 clonal SW 480 cells) divided by 23 cell generations.
Again, a slightly lower rate of variation was obtained by the
subcloning method. Because only one of three randomly selected subclones of SW 480 (i.e., SW 480 R3) differed from
the parental stemline in two clonal chromosomal variations
of the parental karyotype (Table 2), the rate of karyotype
variation according to this method was only 1.4% (33%/
23) per generation.
3.1.3. MDA 231
The karyotypes of 20 cells of a clonal culture of the
breast cancer cell line MDA 231 are shown in Table 3. This
cell line resembled the colon cancer lines as follows: MDA
231 is aneuploid, having a modal chromosome number of
82, and contains 23 clonal marker chromosomes (numbered
M1–M23) and 4 nonclonal markers (numbered M24–M27).
A representative karyotype of MDA 231, prepared with color-coded chromosome-specific probes is shown in Fig. 1E.
As seen from Table 3, 13 out of 20 (65%) MDA 231 cells
had identical karyotypes, and 7 differed from the 13 in one
or more numerical or structural alterations (highlighted in
Table 3). Of the seven variant karyotypes, three differed
from the stemline in a single alteration and four differed
in multiple alterations. Although two of the seven variants
shared one chromosomal alteration (i.e., metaphases 15
and 16; Table 3), they were not grouped together because
they differed in one other chromosomal alteration.
According to the karyotype method, the rate of karyotype variation of MDA 231 was 1.5% per cell generation:
that is, 35% (7 variant cells per 20 clonal MDA cells) divided by 23 clonal cell generations. Based on the subcloning
method, the rate at which the MDA line generates new viable karyotypes was only 0.9% (20%/23) per generation,
because only one out of five subclones of the MDA 231 line
(i.e., MDA R5) differed from the parental stemline in two
new clonal aneusomies (Table 3).
We conclude that the rate at which the three human cancer lines generate new viable karyotype is about 1022 per
cell generation, which is sufficient to explain the high rates
of drug resistance in cancer.
<
Fig. 1. Karyotypes of clonal cultures of the human colon cancer cell lines
HT 29 (A) and SW 480 (C) and of the breast cancer line MDA 231 (E), and
of the puromycin-resistant subclones HT 29 P2 (B), SW 480 P2 (D)m and
MDA 231 P1 (F). The karyotypes were derived from metaphase chromosomes, which were hybridized in situ with color-coded, chromosome-specific DNA probes from MetaSystems (as described in section 2.3). The
numbers identify cytogenetically intact chromosomes and the group labeled ‘‘mar’’ (MetaSystems software) shows chromosomes that are rearranged either intrachromosomally or inter-chromosomally to form
various marker chromosomes. The numbers above these marker chromosomes identify the original chromosomal constituents of hybrid chromosomes in their relative order. N1 and 2 indicate intrachromosomal
amplifications (1) or deletions (2) of the normal chromosome (e.g.,
81). Drug-resistance-specific chromosomal alterations were identified in
part (as discussed in the text) by comparisons of these karyotypes. For example, HT 29 P2 differs from HT 29 in the losses of one copy of chromosomes 6, 11, 21, and M9 191 and the gain of the new hybrid chromosome
M17 11/19/21, and MDA 231 P1 differs from MDA 231 in losses of one
copy of chromosomes 7, 17, and the hybrid chromosome M2 8/12/2 and in
the gain of the new hybrid chromosome M24 2/7.
3.2. Evidence for multiple drug-resistance-specific
chromosomal alterations
To test the prediction of the chromosomal hypothesis, that
drug resistance is generated by resistance-specific chromosomal alterations or aneusomies, we compared the karyotypes of puromycin-resistant subclones of the three human
cancer lines to those of drug-sensitive subclones, which were
randomly selected in the absence of cytotoxic drugs (see sections 2.1 and 2.2). Further, we asked whether independent
drug-resistant clones might share specific chromosomal alterations, reflecting possibly common mechanisms of resistance. Clones with unshared resistance-specific aneusomies
must also be expected, however, because different biochemical mechanisms can generate resistance against the same
drug [4–6,24,32,48,50,57,60,67,86,87]. The results of such
chromosomal analyses of puromycin-resistant clones of
the three lines are described in the sections that follow.
3.2.1. HT 29
As seen from Table 1, three puromycin-resistant subclones of the HT 29 line (i.e., P1–P3) differed from the
parental stemline in six, five, and six clonal chromosomal
alterations, respectively. A representative karyotype of HT
29 P2 is shown in Fig. 1B. HT 29 P2 has lost one copy
of chromosomes 6, 11, 21, and M9 and has gained the
new hybrid chromosome M17 11/19/21. Thus, the average
puromycin-resistant HT 29 clone differed from the parental
stemline in six chromosome alterations (Tables 1 and 4). By
contrast, the average random clone, selected in the absence
of puromycin, differed from the parental stemline in only
0.3 clonal alterations: namely, one clonal aneusomy per
the three clones, R1–R3 (Tables 1 and 4).
Two of the three puromycin-resistant HT 29 clones (i.e.,
HT 29 P2 and P3) shared two clonal alterations compared
to the parental line, the loss of one copy each of the parental chromosomes 11 and 21 (Table 1).
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
49
50
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
3.2.2. SW 480
SW 480 P1 and P2, the two puromycin-resistant subclones of SW 480, differed from the parental stemline in
eight and six clonal alterations, respectively (Table 2). A
slightly exceptional karyotype of SW 480 P2 is shown in
Fig. 1D, because it differed from the P2 clone in the nonclonal (perhaps lethal) absence of chromosome 3 (see Fig. 1D
and Table 2). Thus, the average puromycin-resistant clone
of the SW 480 line differed from the stemline in seven chromosomal alterations (Table 4). By contrast, the average SW
480 clone, randomly selected in the absence of puromycin,
differed from the stemline in only 0.66 chromosomal alterations (two chromosomal alterations per three clones,
R1–R3; Tables 2 and 4).
Both of the puromycin-resistant SW 480 clones shared
two clonal chromosomal alterations compared to the parental line, the loss of one copy each of the parental chromosomes 13 and 17 (Table 2).
3.2.3. MDA 231
MDA 231 P1, one of two puromycin resistant subclones
of MDA 231, differed from the parental line in four new
clonal chromosome alterations (Table 3). A representative
karyotype of MDA 231 P1 is shown in Fig. 1F. By contrast,
five subclones that were randomly selected in the absence
of puromycin differed from the stemline on average in only
0.2 chromosomal alterations: namely, two variations from
one of the five clones, R1–R5 (Tables 3 and 4).
The second puromycin-resistant subclone, MDA 231 P2,
was a near 2n dimer of the parental MDA line, but also differed from it in many chromosomal alterations. For instance, the modal chromosome number of MDA 231 P2
was only 146, compared with the 164 (5 2 82) expected
for a perfect 2n dimer of MDA 231 (Table 3). Thus, this P2
was short by about one to two copies of the chromosomes
3, 6, 8, 9, 10, 13, 14, 16, 17, and M2, compared with a perfectly doubled MDA 231 karyotype (partly shown in Table
3). On the other hand, the P2 had four copies of chromosome 5 and thus two more than expected for a perfect 2n
dimer of the parental MDA line. MDA 231 P2 also contained five new clonal hybrid chromosomes, M25–M29,
of which some were monosomic and others were trisomic
(Table 3). Based on this and on the high number of nonclonal chromosome alterations (highlighted in Table 3), it follows that nonlinear polyploidization had destabilized the
karyotype compared to the parental type. Others have also
observed that nonlinear polyploidization is associated with
increased chromosomal instability [23,54,60]. In view of
uncertainties about the relative losses of chromosomes
compared to a perfect dimer, we have counted only the
six gains of chromosomes of the P2 relative to a hypothetical 2n karyotype of MDA 231 as potentially resistancespecific: namely, the extra pair of chromosome 5 and the
five new clonal marker chromosomes (Table 4).
In sum, the comparisons of the clonal chromosome alterations of puromycin-resistant subclones of the three cancer
cell lines with those of clones selected in the absence of
puromycin and with each other show that the average puromycin-resistant subclone of HT 29, SW 480, and MDA 231
differed from the parental stemline in six clonal chromosomal alterations (see Table 4). By contrast, the average
random subclone, selected in the absence of puromycin,
differed from the parental line in only 0.45 alterations.
Because the drug-resistant clones had been propagated
under our conditions for 8 to 24 generations (see under section 2.2), about the same time as or less than the randomly
selected clones, which were analyzed after about 23 generations (see section 3.1), it follows that most of the extra
aneusomies of resistant clones are resistance-specific.
Others have also shown that in the absence of cytotoxic
drugs the HT 29 line and other human cancer lines are as
stable in culture as we found here [78,88,89]. Moreover,
sharing of some new nonparental chromosomal alterations
by different drug-resistant clones from the same lines, as
in the cases of two puromycin-resistant subclones of HT
29 and SW 480, is also compatible with shared resistance
functions. Thus, our results provide statistical proof that
most resistance-associated aneusomies are resistance-specific. We will present further evidence in the Discussion
section.
4. Discussion
Our results confirm three critical predictions of the chromosomal theory of drug resistance: (i) high rates of resistance compared to mutation via chromosomal variations,
(ii) resistance-specific aneusomies, and (iii) multidrug resistance via unselected genes hitchhiking with chromosomes selected for resistance against specific drugs or for
carcinogenesis.
4.1. High rates of drug resistance via high rates
of chromosomal variations in cancer cells
As predicted, we observed high rates of spontaneous
chromosomal alterations of about 1022 per generation in
three highly aneuploid human cancer cell lines. These rates
are more than sufficient to explain the known rates of drug
resistance in highly aneuploid cancer cells of 1023 to 1025
per generation described previously by others and by us
[2–4,6] (see also section 1. Introduction). The difference
between the rates of drug resistance and the rates of spontaneous chromosome variations observed here would reflect
(i) the lower probabilities of generating resistance-specific
than random aneusomies and (ii) the number of specific variations that seem necessary for the levels of resistance we
have analyzed here. Thus, the inherent instability of aneuploidy can explain both the high rates of spontaneous resistance of cancer cells as well as the high rates of spontaneous
reversion, and even irreversible resistance. Irreversible resistance would reflect a chromosomal cul-de-sac, in which
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
51
Table 2
Karyotypes of a clonal culture of colon cancer line SW480, of a subclonal variant R3, and of two puromycin-resistant subclones, P1 and P2
Metaphases
Clones
Chromosomes
1–6*
SW480
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
M1 2/12
M2 3/12/10
M3 9/1
M4 D9/1
M5 31
M6 8/9
M7 7/14
M8 5/20/7
M9 5/20
M10 D5/20
M11 32
M12 122
M13 19/8/19/5
M14 19/8
M15 15/18
M16 16/14/13
M17 9/5
M18 2/8
M19 9/1/11
M20 12/1
M21 21/11
M8 5/20/7
M9 20/5
2
11
13
14
17
M16 14/9/1
M17 2/11
M18 17/19?
4
13
17
M19 7/9/1
M20 4/9
M21 13/17
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
0
0
0
0
0
0
0
2
1
2
2
1
2
1
2
1
1
2
2
1
1
1
SW480-R3
SW480-P1
SW480-P2
10
15
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
0
1
1
1
1
1
1
1
1
1
1
2
0
0
0
0
0
0
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
0
1
1
1
1
1
1
1
1
1
1
2
0
0
0
0
0
0
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
0
1
1
1
1
1
1
1
1
1
1
2
0
1
0
0
0
0
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
0
0
1
1
1
1
1
1
1
1
1
2
0
0
1
0
1
0
1
2
1
2
1
2
2
1
1
1
3
0
3
2
2
2
3
1
1
2
3
2
2
1
1
1
0
1
1
1
1
1
1
1
1
1
1
2
0
0
0
1
0
1
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
1
1
1
1
0
1
1
1
1
1
1
0
1
1
1
1
0
0
0
0
0
0
1
2
1
2
1
2
2
1
1
1
2
1
3
2
2
2
3
1
1
2
2
2
2
1
1
1
0
1
1
1
1
1
1
1
1
1
1
2
1
0
0
0
0
0
1
2
2
1
2
1
2
1
1
2
2
1
1
1
1
2
2
1
2
1
2
1
1
2
2
1
1
1
1
2
2
1
2
1
2
1
1
2
2
1
1
1
1
2
2
1
2
1
2
1
1
2
2
1
1
1
1
2
2
1
2
1
2
1
1
2
2
1
1
1
1
2
2
1
2
1
2
1
1
2
2
1
1
1
1
2
2
1
2
1
2
1
1
2
2
1
1
1
1
2
2
1
2
1
2
1
1
2
1
1
1
1
0
2
2
1
2
1
2
1
*, identical metaphases; bold, non-clonal aneusomies.
19
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
4
1
1
2
3
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
0
0
0
0
0
0
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
1
3
1
1
2
3
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
0
0
0
0
0
0
1
2
1
2
1
2
2
1
1
1
3
1
3
2
2
2
3
1
1
2
3
2
2
1
1
1
1
1
1
1
1
1
1
2
2
1
1
2
0
0
0
0
0
0
1
2
1
2
1
2
2
1
0
1
3
1
3
2
2
2
3
1
1
2
1
2
2
1
1
1
1
0
1
1
0
1
1
1
1
1
1
2
0
0
0
0
0
0
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
52
Table 3
Karyotypes of a clonal culture of breast cancer line MDA 231, of two puromycin-resistant subclones P1 and P2, and of a random subclone R5
Metaphases
Clones
Chromosomes
1–13*
MDA 231
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
X
M1 21
M2 2/12/8
M3 5/9/1/9/5
M4 7/14/15
M5 1/9/5
M6 7/4/16
M7 4/5
M8 5/20
M9 1/6
M10 2/19/8
M11 1/22
M12 3/18
M13 5/X
M14 15/16
M15 2/X
M16 15/20
M17 8/18
M18 92
M19 112
M20 122
M21 142
M22 9/22
M23 172
M24 2/9/8/5
M25 8/19
M26 11/15
M27 9D
7
17
M2 2/12/8
M24 2/7
2
0
3
2
1
3
2
1
3
4
3
2
3
3
0
2
4
1
4
4
3
2
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
2
3
1
1
0
0
0
0
1
3
1
1
MDA 231-P1
15
2
0
3
2
1
3
2
1
3
4
3
2
3
3
0
2
4
1
4
4
3
2
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
2
3
1
1
0
0
1
0
1
3
1
1
2
0
3
2
1
3
2
1
3
4
3
2
3
3
0
2
4
1
4
4
3
1
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
2
3
1
1
0
0
0
0
1
3
1
1
20
2
0
3
3
1
3
2
1
3
4
3
2
3
3
0
2
4
1
4
4
3
1
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
2
3
1
1
0
0
0
0
1
3
1
1
2
0
3
2
1
3
2
1
3
4
3
2
3
4
0
2
4
1
4
4
3
2
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
2
3
1
1
0
0
0
1
1
3
1
1
2
0
3
2
1
3
2
1
3
4
3
2
3
3
0
2
4
1
4
4
3
2
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
2
3
1
0
0
0
0
0
1
3
1
1
2
0
3
2
1
3
2
1
3
4
3
2
3
3
0
2
4
1
3
4
3
2
1
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
2
0
1
1
2
3
1
1
0
1
0
0
1
4
1
1
2
0
3
2
1
3
2
1
3
4
3
2
3
3
0
2
4
1
4
4
3
2
1
1
2
0
1
1
1
1
1
1
1
0
1
1
1
1
2
1
0
1
2
3
2
1
1
0
0
0
1
4
1
1
(Continued)
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
53
Table 3
(Continued)
Metaphases
1
MDA 231-P2**
MDA 231-R5
3
5
6
8
9
10
13
14
16
17
M2 2/12/8
M25 141
M26 7/20
M27 7/17
M28 14/15
M29 9/26
M16 15/20
M21 142
4
4
5
1
4
6
6
5
3
6
3
1
1
3
1
1
1
2
5
4
4
5
1
4
6
5
5
3
6
3
1
1
3
1
0
1
2
4
4
5
1
4
6
5
5
3
6
2
1
1
3
1
1
1
2
4
4
5
1
4
6
5
5
3
6
3
1
1
3
0
1
1
2
4
4
5
1
4
6
5
4
3
6
3
1
1
3
1
1
1
2
10
4
4
5
1
5
6
5
4
3
6
3
1
1
3
1
1
1
2
4
4
5
1
4
6
5
4
3
5
3
1
1
3
0
1
1
2
4
3
5
1
5
5
4
4
3
3
2
1
1
2
1
1
1
2
4
4
5
1
6
6
4
2
3
3
3
2
2
3
0
1
1
2
3
4
5
0
4
5
5
4
3
7
2
2
1
2
1
1
1
1
7
5
10
1
5
8
7
5
6
5
3
2
1
3
2
1
*, identical metaphases; **, remaining metaphases near 2n of MDA 231; bold, non-clonal aneusomies.
the loss or further alteration of a resistance-specific chromosome is lethal. The chromosomal theory also explains intrinsic drug resistance of previously untreated cancer cells
[24,34,50] by preexisting cancer-specific chromosomal
alterations, which accidentally include resistance-specific
functions.
4.2. Resistance-specific chromosomal alterations
Because drug-resistant subclones of the cancer cells tested here carried on average O10 times more new aneusomies than randomly selected clones and shared several of
these alterations, the odds are negligible that these chromosomal associations with drug resistance are by chance
(Table 4). Recent evidence, that the expression of the thousands of genes that are associated with resistance-specific
chromosome alterations is proportional to the altered ploidy
of the respective chromosomes, lends functional support to
the view that resistance is dependent on specific
Table 4
New clonal aneusomies associated with puromycin-resistant (P)
and randomly selected nonresistant (R) subclones of the human colon
and breast cancer cell lines HT 29, SW 480, and MDA 231
Clonal aneusomies, no.
P subclones
New per clone
Average
per clone
New shared
R subclones
New per clone
Average
per clone
HT 29
SW 480
MDA 231
Totals
P1, P2, P3
6, 5, 6
~6
P1, P2
8, 6
7
P1, P2
4, 6
5
41
~6
2/P2 & P3
R1, R2, R3
0, 0, 1
0.33
2/P1 & P2
R1, R2, R3
0, 0, 2
0.66
?
4/41
R1, R2, R3, R4, R5
0, 0, 0, 0, 2
5
0.2
0.45
Data are taken from Tables 1–3.
aneusomies [49,50,67,71,72]. Most important, we have
found here that the degree of resistance is proportional to
the numbers of resistance-associated karyotype alterations
(preliminary results obtained in the stepwise selection of resistance as described in section 2.2), which agrees with
similar results obtained previously by others [28,37,49].
Thus, the stepwise selection of drug resistance [5] appears
to correspond to the accumulation of resistance specific
aneusomiesdjust as multistep carcinogenesis corresponds
to the accumulation of cancer-specific aneusomies [90].
Therefore, we conclude that most resistance-associated
aneusomies are resistance-specific.
Nonetheless, neither we nor others have yet been able to
define how each of the diverse chromosomal alterations that
are not shared but that are associated with resistance against
the same drug may generate the same kind of resistance or
resistances. The literature provides examples of structurally
nonidentical aneusomies generating the same resistances
[5,6,21–24,30,32,36,38,42–62,67,86,87]. A probable reason
is the existence of known multiple pathways generating the
same resistance [4–6,24,32,48,50,57,60,67,86,87]. For example, Tlsty named at least three alternative mechanisms
for resistance against methotrexate, ‘‘altered transport, altered enzyme (folate reductase), and amplification,’’ [6]
and Wang et al. [67] and Rooney et al. [55] showed chromosomally different patterns of fluorouracil resistance. Again,
the fact that the same types of cancers also share only partially specific or nonrandom cancer-specific aneusomies
may serve as a model [79,83,91–95].
4.3. Multidrug resistance
Mutational theories have failed to explain how singular
cytotoxic drugs induce multidrug resistance in cancer cells
54
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
(see section 1. Introduction) and how cancer cells from
which multiple, hypothetical drug-resistance genes have
been deleted still become multidrug resistant [4]. In contrast, the chromosomal theory proposes that multidrug resistance reflects functions of some of the thousands of
unselected genes that hitchhike with chromosomes selected
for resistance against singular drugs. Likewise, the theory
explains intrinsic drug resistance by resistance-specific
genes among the thousands of unselected genes that are accidentally associated with cancer-specific aneusomies.
We conclude that the high rates of drug resistance in
cancer cells reflect aneuploidy-catalyzed generation of resistance-specific aneusomies, and that multidrug resistance
reflects functions of the thousands of unselected genes that
hitchhike with chromosomes selected for resistance against
specific drugs. Thus, aneuploidy is the primary cause of
drug resistance in cancer cells.
Earlier studies on chromosomal theories of drug resistance support this conclusion. For example, in the 1950s
and 1960s Vogt [21] and Biedler et al. [96] provided correlative evidence for a chromosomal basis of drug resistance.
In 1974, Terzi [30] proposed that, in the absence of mutagens, ‘‘chromosomal variation’’ would be ‘‘the origin of
drug-resistant mutants.’’ Most recently, Doubre et al. [34]
have proposed that multidrug resistance ‘‘could result from
chromosome reassortments catalyzed by aneuploidy.’’
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Acknowledgment
We thank Harvey Bialy (Institute of Biotechnology, Autonomous National University of Mexico–UNAM, Cuernavaca), Athel Cornish-Bowden (CNRS, University of
Marseille, France), George Miklos (Human Genetic Signatures and GenetixXpress, Sydney, Australia), and Brandt
Schneider (Texas Tech University Health Sciences Center,
Lubbock, TX) for critical reviews of and helpful suggestions for the manuscript. David Rasnick (Pretoria, South
Africa) is thanked for valuable information. We are indebted to the Abraham J. and Phyllis Katz Foundation (New
York, NY), Robert Leppo (philanthropist, San Francisco,
CA), an American foundation that prefers to be anonymous, other private sources, and the Forschungsfonds der
Fakultaet fuer Klinische Medizin Mannheim for support.
Robert Leppo is specifically acknowledged for the gift of
the Zeiss microscope equipped with fluorescence optics
for mFISH karyotyping. P.D. is grateful to the Deutsche
Krebshilfe for a guest professorship at Mannheim.
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
References
[27]
[1] Law LW. Origin of the resistance of leukaemic cells to folic acid
antagonists. Nature 1952;169:628–9.
[2] Harris H. The cells of the body; a history of somatic cell genetics.
Plainview, NY: Cold Spring Harbor Laboratory Press, 1995.
[3] Duesberg P, Stindl R, Hehlmann R. Explaining the high mutation
rates of cancer cells to drug and multidrug resistance by chromosome
[28]
reassortments that are catalyzed by aneuploidy. Proc Natl Acad Sci
U S A 2000;97:14295–300.
Duesberg P, Stindl R, Hehlmann R. Origin of multidrug resistance in
cells with and without multidrug resistance genes: chromosome reassortments catalyzed by aneuploidy. Proc Natl Acad Sci U S A 2001;
98:11283–8.
Schoenlein PV. Molecular cytogenetics of multiple drug resistance.
Cytotechnology 1993;12:63–89.
Tlsty TD. Normal diploid human and rodent cells lack a detectable
frequency of gene amplification. Proc Natl Acad Sci U S A
1990;87:3132–6.
Lewin B. Genes VI. Oxford: Oxford University Press, 1997.
Vogel F, Motulsky AG. Human genetics: problems and approaches.
Berlin: Springer Verlag, 1986.
Marx J. Debate surges over the origins of genomic defects in cancer.
Science 2002;297:544–6.
Holliday R. Neoplastic transformation: the contrasting stability of
human and mouse cells. In: Lindahl T, Tooze J, editors. Genetic
instability in cancer. Plainview, NY: Cold Spring Harbor Laboratory
Press, 1996, pp. 103–15.
Oshimura M, Barrett JC. Chemically induced aneuploidy in mammalian cells: mechanisms and biological significance in cancer. Environ
Mutagen 1986;8:129–59.
Sieber OM, Heinimann K, Tomlinson IP. Genomic instability:
the engine of tumorigenesis? Nat Rev Cancer 2003;3:701–8.
Tomlinson IP, Novelli MR, Bodmer WF. The mutation rate and
cancer. Proc Natl Acad Sci U S A 1996;93:14800–3.
Strauss BS. The origin of point mutations in human tumor cells.
Cancer Res 1992;52:249–53.
Harris CC. Chemical and physical carcinogenesis: advances and
perspective for the 1990s. Cancer Res 1991;51:5023s–44s.
Duesberg P, Fabarius A, Hehlmann R. Aneuploidy, the primary cause
of the multilateral genomic instability of neoplastic and preneoplastic
cells. IUBMB Life 2004;56:65–81.
Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in
human cancers. Nature 1998;396:643–9.
Wang TL, Rago C, Silliman N, Ptak J, Markowitz S, Willson JK,
Parmigiani G, Kinzler KW, Vogelstein B, Velculescu VE. Prevalence
of somatic alterations in the colorectal cancer cell genome. Proc Natl
Acad Sci U S A 2002;99:3076–80.
Littlefield JW. Studies on thymidine kinase in cultured mouse fibroblasts. Biochim Biophys Acta 1965;95:14–22.
Lieberman I, Ove P. Isolation and study of mutants from mammalian
cells in culture. Proc Natl Acad Sci U S A 1959;45:867–77.
Vogt M. A study of the relationship between karyotype and phenotype in cloned lines of strain HeLa. Genetics 1959;44:1257–70.
Biedler JL, Riehm H. Cellular resistance to actinomycin D in Chinese
hamster cells in vitro: cross-resistance, radioautographic, and cytogenetic studies. Cancer Res 1970;30:1174–84.
Slovak ML, Hoeltge GA, Trent JM. Cytogenetic alterations associated with the acquisition of doxorubicin resistance: possible
significance of chromosome 7 alterations. Cancer Res 1987;47:
6646–52.
Goldie JH. Drug resistance in cancer: a perspective. Cancer Metastasis Rev 2001;20:63–8.
Biedler JL, Albrecht AM, Hutchison DJ, Spengler BA. Drug response, dihydrofolate reductase, and cytogenetics of amethopterinresistant Chinese hamster cells in vitro. Cancer Res 1972;32:153–61.
Schimke RT. Gene amplification, drug resistance, and cancer. Cancer
Res 1984;44:1735–42.
Tlsty TD, Margolin BH, Lum K. Differences in the rates of gene amplification in nontumorigenic and tumorigenic cell lines as measured
by Luria-Delbrueck fluctuation analysis. Proc Natl Acad Sci U S A
1989;86:9441–5.
Howell N, Belli TA, Zaczkiewicz LT, Belli JA. High-level, unstable
adriamycin resistance in a Chinese hamster mutant cell line with
double minute chromosomes. Cancer Res 1984;44:4023–9.
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
[29] Gartler SM, Pious DE. Genetics of mammalian cell cultures. Humangenetik 1966;2:83–114.
[30] Terzi M. Chromosomal variation and the origin of drug-resistant
mutants in mammalian cell lines. Proc Natl Acad Sci U S A
1974;71:5027–31.
[31] Pauletti G, Lai E, Attardi G. Early appearance and long-term persistence of the submicroscopic extrachromosomal elements (amplisomes) containing the amplified DHFR genes in human cancer cell
lines. Proc Natl Acad Sci U S A 1990;87:2955–9.
[32] Schimke RT. Gene amplification in cultured animal cells. Cell 1984;
37:705–13.
[33] Chabner BA, Roberts TG. Timeline: chemotherapy and the war on
cancer. Nat Rev Cancer 2005;5:65–72.
[34] Doubre H, Cesari D, Mairovitz A, Benac C, Chantot-Bastaraud S,
Dagnon K, Antoine M, Danel C, Bernaudin JF, Fleury-Feith J.
Multidrug resistance-associated protein (MRP1) is overexpressed in
DNA aneuploid carcinomatous cells in non-small cell lung cancer
(NSCLC). Int J Cancer 2005;113:568–74.
[35] DeVita VT Jr. Principles of chemotherapy. In: DeVita VT Jr,
Hellman S, Rosenberg SA, editors. Cancer: principles and practice
of oncology. Philadelphia: Lippincott, 1993, pp. 276–92.
[36] Nieuwint AW, Baas F, Wiegant J, Joenje H. Cytogenetic alterations
associated with P-glycoprotein- and non-P-glycoprotein-mediated
multidrug resistance in SW-1573 human lung tumor cell lines.
Cancer Res 1992;52:4361–71.
[37] Dahllof B, Martinsson T, Levan G. Resistance to actinomycin D and
to vincristine induced in a SEWA mouse tumor cell line with concomitant appearance of double minutes and a low molecular weight
protein. Exp Cell Res 1984;152:415–26.
[38] Fojo AT, Whang-Peng J, Gottesman MM, Pastan I. Amplification of
DNA sequences in human multidrug-resistant KB carcinoma cells.
Proc Natl Acad Sci U S A 1985;82:7661–5.
[39] Meyers MB, Spengler BA, Chang TD, Melera PW, Biedler JL. Gene
amplification-associated cytogenetic aberrations and protein changes
in vincristine-resistant Chinese hamster, mouse, and human cells.
J Cell Biol 1985;100:588–97.
[40] Roy SN, Horwitz SB. A phosphoglycoprotein associated with taxol
resistance in J774.2 cells. Cancer Res 1985;45:3856–63.
[41] Gottesman MM, Hrycyna CA, Schoenlein PV, Germann UA,
Pastan I. Genetic analysis of the multidrug transporter. Annu Rev
Genetics 1995;29:607–49.
[42] Chu EHY, Giles NH. Comparative chromosomal studies on mammalian cells in culture: I. The HeLa strain and its mutant clonal derivatives. J Natl Cancer Inst 1958;20:383–401.
[43] Hsu TC. Chromosomal evolution in cell populations. Int Rev Cytol
1961;12:69–161.
[44] Tew KD, Moy BC, Hartley-Asp B. Acquired drug resistance is accompanied by modification in the karyotype and nuclear matrix of
a rat carcinoma cell line. Exp Cell Res 1983;149:443–50.
[45] Scheck AC, Mehta BM, Beikman MK, Shapiro JR. BCNU-resistant
human glioma cells with over-representation of chromosomes
7 and 22 demonstrate increased copy number and expression of platelet-derived growth factor genes. Genes Chromosomes Cancer 1993;8:
137–48.
[46] Leyland-Jones B, Kelland LR, Harrap KR, Hiorns LR. Genomic imbalances associated with acquired resistance to platinum analogues.
Am J Pathol 1999;155:77–84.
[47] Wasenius VM, Jekunen A, Monni O, Joensuu H, Aebi S, Howell SB,
Knuutila S. Comparative genomic hybridization analysis of chromosomal changes occurring during development of acquired resistance
to cisplatin in human ovarian carcinoma cells. Genes Chromosomes
Cancer 1997;18:286–91.
[48] Rao PH, Houldsworth J, Palanisamy N, Murty VV, Reuter VE,
Motzer RJ, Bosl GJ, Chaganti RS. Chromosomal amplification is associated with cisplatin resistance of human male germ cell tumors.
Cancer Res 1998;58:4260–3.
55
[49] Koski T, Karhu R, Visakorpi T, Vilpo L, Knuutila S, Vilpo J. Complex chromosomal aberrations in chronic lymphocytic leukemia are
associated with cellular drug and irradiation resistance. Eur J Haematol 2000;65:32–9.
[50] Yasui K, Mihara S, Zhao C, Okamoto H, Saito-Ohara F, Tomida A,
Funato T, Yokomizo A, Naito S, Imoto I, Tsuruo T, Inazawa J. Alteration in copy numbers of genes as a mechanism for acquired drug
resistance. Cancer Res 2004;64:1403–10.
[51] Kotchetkov R, Cinatl J, Blaheta R, Vogel JU, Karaskova J, Squire J,
Hernaiz Driever P, Klingebiel T, Cinatl J Jr. Development of
resistance to vincristine and doxorubicin in neuroblastoma alters
malignant properties and induces additional karyotype changes: a
preclinical model. Int J Cancer 2003;104:36–43.
[52] Struski S, Cornillet-Lefebvre P, Doco-Fenzy M, Dufer J, Ulrich E,
Masson L, Michel N, Gruson N, Potron G. Cytogenetic characterization of chromosomal rearrangement in a human vinblastine-resistant
CEM cell line: use of comparative genomic hybridization and fluorescence in situ hybridization. Cancer Genet Cytogenet 2002;132:51–4.
[53] Hattinger CM, Reverter-Branchat G, Remondini D, Castellani GC,
Benini S, Pasello M, Manara MC, Scotlandi K, Picci P, Serra M. Genomic imbalances associated with methotrexate resistance in human
osteosarcoma cell lines detected by comparative genomic hybridization-based techniques. Eur J Cell Biol 2003;82:483–93.
[54] Pittman SM, Kavallaris M, Stewart BW. Karyotypic analysis of
CCRF-CEM and drug-resistant cell lines with stable and unstable
ploidy. Cancer Genet Cytogenet 1993;66:54–62.
[55] Rooney PH, Stevenson DA, Marsh S, Johnston PG, Haites NE,
Cassidy J, McLeod HL. Comparative genomic hybridization analysis
of chromosomal alterations induced by the development of resistance
to thymidylate synthase inhibitors. Cancer Res 1998;58:5042–5.
[56] Chen GK, Lacayo NJ, Duran GE, Wang Y, Bangs CD, Rea S,
Kovacs M, Cherry AM, Brown JM, Sikic BI. Preferential expression
of a mutant allele of the amplified MDR1 (ABCB1) gene in drugresistant variants of a human sarcoma. Genes Chromosomes Cancer
2002;34:372–83.
[57] Achuthan R, Bell SM, Roberts P, Leek JP, Horgan K, Markham AF,
MacLennan KA, Speirs V. Genetic events during the transformation
of a tamoxifen-sensitive human breast cancer cell line into a drugresistant clone. Cancer Genet Cytogenet 2001;130:166–72.
[58] Weise A, Liehr T, Efferth T, Kuechler A, Gebhart E. Comparative MFISH and CGH analyses in sensitive and drug-resistant human T-cell
acute leukemia cell lines. Cytogenet Genome Res 2002;98:118–25.
[59] Shimizu H, Fukuda T, Ghazizadeh M, Nagashima M, Kawanami O,
Suzuki T. Molecular cytogenetic characterization of drug-resistant
leukemia cell lines by comparative genomic hybridization and fluorescence in situ hybridization. Jpn J Cancer Res 2002;93:902–10.
[60] Struski S, Doco-Fenzy M, Koehler M, Chudoba I, Levy F, Masson L,
Michel N, Ulrich E, Gruson N, Benard J, Potron G, CornilletLefebvre P. Cytogenetic evolution of human ovarian cell lines associated with chemoresistance and loss of tumorigenicity. Anal Cell
Pathol 2003;25:115–22.
[61] Knutsen T, Rao VK, Ried T, Mickley L, Schneider E, Miyake K,
Ghadimi BM, Padilla-Nash H, Pack S, Greenberger L, Cowan K,
Dean M, Fojo T, Bates S. Amplification of 4q21-q22 and the MXR
gene in independently derived mitoxantrone-resistant cell lines.
Genes Chromosomes Cancer 2000;27:110–6.
[62] Kudoh K, Takano M, Koshikawa T, Hirai M, Yoshida S, Mano Y,
Yamamoto K, Ishii K, Kita T, Kikuchi Y, Nagata I, Miwa M,
Uchida K. Gains of 1q21-q22 and 13q12-q14 are potential indicators
for resistance to cisplatin-based chemotherapy in ovarian cancer patients. Clin Cancer Res 1999;5:2526–31.
[63] Tlsty TD. Genomic instability and its role in neoplasia. In:
Kastan MB, editor. Genetic instability and tumorigenesis. Berlin:
Springer, 1997, pp. 37–46.
[64] Singer MJ, Mesner LD, Friedman CL, Trask BJ, Hamlin JL. Amplification of the human dihydrofolate reductase gene via double
56
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
R. Li et al. / Cancer Genetics and Cytogenetics 163 (2005) 44–56
minutes is initiated by chromosome breaks. Proc Natl Acad Sci
U S A 2000;97:7921–6.
Siminovitch L. On the nature of heritable variation in cultured
somatic cells. Cell 1976;7:1–11.
Szybalski W. Genetics of human cell lines: II. Method for determination of mutation rates to drug resistance. Exp Cell Res 1960;18:
588–91.
Wang TL, Diaz LA Jr, Romans K, Bardelli A, Saha S, Galizia G,
Choti M, Donehower R, Parmigiani G, Shih IeM, IacobuzioDonahue C, Kinzler KW, Vogelstein B, Lengauer C, Velculescu VE.
Digital karyotyping identifies thymidylate synthase amplification as
a mechanism of resistance to 5-fluorouracil in metastatic colorectal
cancer patients. Proc Natl Acad Sci U S A 2004;101:3089–94.
Duesberg P, Rausch C, Rasnick D, Hehlmann R. Genetic instability
of cancer cells is proportional to their degree of aneuploidy. Proc Natl
Acad Sci U S A 1998;95:13692–7.
Fabarius A, Hehlmann R, Duesberg PH. Instability of chromosome
structure in cancer cells increases exponentially with degrees of
aneuploidy. Cancer Genet Cytogenet 2003;143:59–72.
Richardson C, Jasin M. Frequent chromosomal translocations
induced by DNA double-strand breaks. Nature 2000;405:697–700.
van ’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M,
Peterse HL, van der Kooy K, Marton MJ, Witteveen AT,
Schreiber GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R,
Friend SH. Gene expression profiling predicts clinical outcome of
breast cancer. Nature 2002;415:530–6.
O’Neill GM, Catchpoole DR, Golemis EA. From correlation to
causality: microarrays, cancer, and cancer treatment. Biotechniques
2003;Mar(Suppl):64–71.
Li R, Rasnick D, Duesberg P. Correspondence re: D. Zimonjic et al.,
Derivation of human tumor cells in vitro without widespread genomic
instability: Cancer Res., 61:8838–8844, 2001. Cancer Res 2002;62:
6345–8. author reply 6348–9 [Erratum in: Cancer Res 2002;62:7380].
Duesberg P, Stindl R, Li R, Hehlmann R, Rasnick D. Aneuploidy
versus gene mutation as cause of cancer. Curr Sci 2001;81:490–500.
Daza P, Torreblanca J, Moreno FJ. The comet assay differentiates
efficiently and rapidly between genotoxins and cytotoxins in quiescent cells. Cell Biol Int 2004;28:497–502.
Budavari S, editor. The Merck index: an encyclopedia of chemicals,
drugs, biologicals. 11th ed. Rahway, NJ: Merck, 1989.
Yoon DS, Wersto RP, Zhou W, Chrest FJ, Garrett ES, Kwon TK,
Gabrielson E. Variable levels of chromosomal instability and mitotic
spindle checkpoint defects in breast cancer. Am J Pathol 2002;161:
391–7.
Kawai K, Viars C, Arden K, Tarin D, Urquidi V, Goodison S. Comprehensive karyotyping of the HT-29 colon adenocarcinoma cell line.
Genes Chromosomes Cancer 2002;34:1–8.
Heim S, Mitelman F. Cancer cytogenetics. 2nd ed. New York: WileyLiss, 1995.
Sandberg AA. The chromosomes in human cancer and leukemia.
2nd ed. New York: Elsevier Science, 1990.
[81] Mayer VW, Aguilera A. High levels of chromosome instability in polyploids of Saccharomyces cerevisiae. Mutat Res 1990;231:177–86.
[82] Levan A, Biesele JJ. Role of chromosomes in cancerogenesis, as
studied in serial tissue culture of mammalian cells. Ann N Y Acad
Sci 1958;71:1022–53.
[83] Koller PC. The role of chromosomes in cancer biology. New York:
Springer-Verlag, 1972.
[84] Giaretti W. A model of DNA aneuploidization and evolution in colorectal cancer. Lab Invest 1994;71:904–10.
[85] Shackney SE, Berg G, Simon SR, Cohen J, Amina S,
Pommersheim W, Yakulis R, Wang S, Uhl M, Smith CA,
Pollice A, Hartsock R. Origins and clinical implications of aneuploidy in early bladder cancer. Cytometry 1995;22:307–16.
[86] Albrecht AM, Biedler JL, Hutchison DJ. Two different species of dihydrofolate reductase in mammalian cells differentially resistant to
amethopterin and methasquin. Cancer Res 1972;32:1539–46.
[87] Orkin SH, Littlefield JW. Mutagenesis to aminopterin resistance in
cultured hamster cells. Exp Cell Res 1971;69:174–80.
[88] Roschke AV, Stover K, Tonon G, Schaffer AA, Kirsch IR. Stable karyotypes in epithelial cancer cell lines despite high rates of ongoing
structural and numerical chromosomal instability. Neoplasia 2002;
4:19–31.
[89] Roschke AV, Tonon G, Gehlhaus KS, McTyre N, Bussey KJ,
Lababidi S, Scudiero DA, Weinstein JN, Kirsch IR. Karyotypic complexity of the NCI-60 drug-screening panel. Cancer Res 2003;63:
8634–47.
[90] Duesberg P, Li R. Multistep carcinogenesis: a chain reaction of aneuploidizations. Cell Cycle 2003;2:202–10.
[91] Hoglund M, Gisselsson D, Mandahl N, Johansson B, Mertens F,
Mitelman F, Sall T. Multivariate analyses of genomic imbalances in
solid tumors reveal distinct and converging pathways of karyotypic
evolution. Genes Chromosomes Cancer 2001;31:156–71.
[92] Ried T, Heselmeyer-Haddad K, Blegen H, Schrock E, Auer G. Genomic changes defining the genesis, progression, and malignancy potential in solid human tumors: a phenotype/genotype correlation. Genes
Chromosomes Cancer 1999;25:195–204.
[93] Fabarius A, Willer A, Yerganian G, Hehlmann R, Duesberg P.
Specific aneusomies in Chinese hamster cells at different stages of
neoplastic transformation, initiated by nitrosomethylurea. Proc Natl
Acad Sci U S A 2002;99:6778–83.
[94] Balaban GB, Herlyn M, Clark WH Jr, Nowell PC. Karyotypic evolution in human malignant melanoma. Cancer Genet Cytogenet
1986;19:113–22.
[95] Wilkens L, Flemming P, Gebel M, Bleck J, Terkamp C, Wingen L,
Kreipe H, Schlegelberger B. Induction of aneuploidy by increasing
chromosomal instability during dedifferentiation of hepatocellular
carcinoma. Proc Natl Acad Sci U S A 2004;101:1309–14.
[96] Biedler JL, Albrecht AM, Hutchison DJ. Cytogenetics of mouse leukemia L1210: I. Association of a specific chromosome with dihydrofolate reductase activity in amethopterin-treated sublines. Cancer Res
1965;25:246–57.