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Drug Resistance Updates xxx (2007) xxx–xxx
Cancer drug resistance: The central role of the karyotype夽
Peter Duesberg a,∗ , Ruhong Li a , Rainer Sachs b , Alice Fabarius c ,
Madhvi B. Upender d , Ruediger Hehlmann c
b
a Dept. Mol. and Cell Biol., Donner Laboratory, UC Berkeley, Berkeley, CA 94720, USA
Departments of Mathematics and Physics, Evans Hall MC3840, UC Berkeley, Berkeley, CA 94720, USA
c III. Medizinische Klinik Mannheim of the University of Heidelberg at Mannheim,
Wiesbadener Str. 7-11, 68305 Mannheim, Germany
d Genetics Branch, Center for Cancer Research, National Cancer Institute/NIH, Building 50,
50 South Drive, Bethesda, MD 20892-8010, USA
Received 23 October 2006; received in revised form 14 December 2006; accepted 5 February 2007
Abstract
Current genetic and epigenetic theories of cancer-specific drug resistance do not adequately explain: (i) the karyotypic changes that coincide
with resistance, (ii) the high rates at which cancer cells acquire and enhance resistance compared to the rates of conventional mutation, (iii) the
wide ranges of resistance such as multidrug resistance, (iv) the frequent occurrence of intrinsic drug resistance. We have recently proposed, that
specific karyotypic alterations are sufficient for drug resistance via new transcriptomes of cooperative genes, independent of gene mutation.
This mechanism generates new phenotypes just like trisomy 21 generates Down syndrome. These karyotypic changes are generated by
cancer-specific aneuploidy autocatalytically, because aneuploidy destabilizes the karyotype by misbalancing teams of proteins that synthesize,
repair and segregate chromosomes. Evidence for this chromosomal mechanism is as follows: (i) resistance is proportional to the number of
clonal chromosomal alterations compared to drug-sensitive precursors. (ii) The high rates at which cancer cells acquire drug resistance are
comparable with the rates, as high as 10−2 per cell generation, at which their karyotypes change—dimming hopes for gene-specific therapies.
(iii) Multidrug resistance probably reflects un-selected transcriptomes of karyotypes selected for resistance against specific drugs. (iv) Intrinsic
drug resistance probably reflects unselected transcriptomes of karyotypes selected for oncogenicity. We also adduce evidence that resistance
of chronic myeloid leukemia against the drug imatinib is chromosomal, although it is widely believed to be due to mutation of a kinase.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Drug resistance; Cancer; Karyotype; Aneuploidy; Mutations; Imatinib
1. Introduction
Cancer cells rapidly acquire resistance against numerous
cytotoxic drugs or are even intrinsically resistant (Chabner
and Roberts, 2005; Goldie, 2001; O’Driscoll and Clynes,
2006). By contrast, normal cells – in particular those of cancer patients – remain drug-sensitive even under prolonged
treatment (Duesberg et al., 2000, 2001; Tlsty, 1990; Wright
et al., 1990).
夽 This paper is the first part of a Debate on genetic and epigenetic causes
of drug resistance in cancer.
∗ Corresponding author. Tel.: +1 510 642 6549; fax: +1 510 643 6455.
E-mail address: [email protected] (P. Duesberg).
Despite over 50 years of research (Chabner and Roberts,
2005; Law, 1952), it is still debated, how cancer cells
generate complex resistance phenotypes against a multitude
of drugs much more rapidly than predicted by conventional
mutation, and how cancer cells generate such resistance with
the very same genes they just inherited from normal, drugsensitive precursor cells (Alaoui-Jamali et al., 2004; Goldie,
2001; Li et al., 2005; McDonald et al., 2005; Richardson
and Kaye, 2005; Schoenlein, 1993). This paradox has been
termed the “differential utilization of the genome” by cancer
cells (Foulds, 1969).
The search for causes of cancer-specific drug resistance
is currently focused on gene mutations (genetic theory), and
non-mutational alterations of gene functions (epigenetic theory) (Chen et al., 2005; Goldie, 2001; Michor et al., 2006;
1368-7646/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.drup.2007.02.003
Please cite this article in press as: Duesberg, P. et al., Cancer drug resistance: The central role of the karyotype, Drug Resist. Updat. (2007),
doi:10.1016/j.drup.2007.02.003
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Morgillo and Lee, 2005; Richardson and Kaye, 2005; Roberti
et al., 2006; Schoenlein, 1993). In our opinion, neither theory provides adequate explanations for four landmarks of
cancer-specific drug resistance, discussed below.
prior random change followed by selection as opposed
to induction” (Goldie, 2001). Moreover, the instability of
drug resistance of many cancer cells differs from epigenetic
alterations of developing normal cells, which are often stable
for the lifetime of an organism and are even conserved in
cancer cells (Ross et al., 2000).
2. Landmarks of cancer-specific drug resistance
2.1. Cancer-specific drug resistance coincides with
specific karyotype alterations
Cancer cells differ from normal cells in clonal and
non-clonal karyotypic alterations (Duesberg et al., 2005;
Fabarius et al., 2002; Heim and Mitelman, 1995; Heselmeyer
et al., 1997; Tsafrir et al., 2006). When cancer cells acquire
resistance against drugs, they acquire new karyotypic alterations and/or they lose old ones (Kotchetkov et al., 2005; Li
et al., 2005; McDonald et al., 2005; Terzi, 1974; Vogt, 1959).
By contrast, conventional mutations and developmental
epigenetic alterations do not coincide with alterations of the
karyotype.
2.2. Cancer cells acquire drug resistance at very high
rates compared to mutation or epigenetic alteration
In the presence of cytotoxic drugs cancer cells become
spontaneously resistant at rates of up to 10−3 per cell generation, based on karyotypic changes at rates of 10−2 (see
Section 4.2 below) (Law, 1952; Li et al., 2005; Schoenlein,
1993; Tlsty, 1990). Typically, drug resistance is acquired
at these high rates in multiple steps, generating levels of
resistance up to 105 -fold higher than sensitive cells (Law,
1952; Schoenlein, 1993; Vogt, 1959). Conversely, most cancer cell populations lose drug resistance in the absence of
cytotoxic drugs at the same high rates at which they acquired
it (Duesberg et al., 2000, 2001; Schimke, 1984; Schoenlein,
1993; Tlsty, 1990).
By contrast, the rates of conventional mutation in normal diploid cells are only around 10−7 per mono-allelic and
10−14 per bi-allelic gene per cell generation (Lewin, 1997;
Vogel and Motulsky, 1986). The same low mutation rates
also apply to >90% of cancer cells (Lengauer et al., 1998;
Marx, 2002; Tomlinson et al., 1996; Wang et al., 2002).
Thus the rates at which cancer cells become drug resistant
or lose resistance are 4–11 orders higher than conventional
mutation rates. These exceptionally high rates have thus been
called “intrinsic mutability” to signal as yet unknown cancerspecific mechanisms (Chabner and Roberts, 2005).
The rapid development of drug resistance in cancer
cells is also incompatible with epigenetic alterations of
developing normal cells. Since “the development of clinical
resistance sometimes occurs so rapidly, it is tempting to
assume that this must represent some type of directly induced
event” (Goldie, 2001). But, “when rigorous experiments are
performed to distinguish between mutation and inducible
epigenetic mechanisms . . . by using . . . the Luria-Delbrueck
fluctuation test . . . the results are always consistent with
2.3. Resistance to one specific drug is acquired together
with resistance to many unrelated drugs and with altered
malignancy or morphology
Numerous studies show that cancer cells typically acquire
resistance against one specific drug together with resistance against many unrelated drugs—as multidrug resistance
(Alaoui-Jamali et al., 2004; Duesberg et al., 2001; O’Driscoll
and Clynes, 2006; Schoenlein, 1993). In addition, drug resistance is often acquired together with increased malignancy
(Kotchetkov et al., 2005) and altered morphology (Duesberg
et al., 2000). In view of this “complexity and heterogeneity” (Alaoui-Jamali et al., 2004; McDonald et al., 2005),
cancer-specific drug resistance has been described as “multifactorial” (O’Driscoll and Clynes, 2006), involving “multiple
pathways” (Richardson and Kaye, 2005). The simultaneous
acquisition of resistance against multiple drugs and altered
malignancy or morphology by mutation, however, would
typically require multiple independent mutations—a highly
improbable scenario.
Nevertheless, several human and murine genes have
already been named multidrug resistance genes (Schoenlein,
1993). But, experimental tests from our lab have shown that
aneuploid cells of mice from which all known, putatively multidrug resistance genes, mdr1a, mdr1b and mrp1, had been
deleted (Allen et al., 2000), still become multidrug resistant
(Duesberg et al., 2001).
During development, epigenetic mechanisms are known
to change multiple phenotypic traits at once. But there is
no precedent either for the activation of putative multidrug
resistance genes in normal development nor for the linkage
between epigenetic activation and karyotypic alterations, as
are found in cancer cells.
2.4. Intrinsic drug resistance of cancer cells
Many cancers are drug- and even multidrug resistant
intrinsically, prior to exposure to any cytotoxic drugs (Goldie,
2001; McDonald et al., 2005; O’Driscoll and Clynes, 2006).
However, there is no genetic or epigenetic precedent for the
inheritance of drug or multidrug resistance by non-cancer
cells.
3. The karyotypic theory of cancer-specific drug
resistance
Since drug-resistant cancer cells differ from drug sensitive progenitors in karyotypic alterations and since most
cellular phenotypes are produced by multigenic transcrip-
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tomes (Beadle and Tatum, 1941; Hartman et al., 2001;
Kacser and Burns, 1981), we have recently proposed a new
karyotypic theory of drug resistance. This theory postulates
that specific chromosomal alterations cause drug resistance
by altering the stoichiometry and integrity of multigenic
transcriptomes—just as trisomy 21 causes Down syndrome
(Li et al., 2005). Owing to the many transcriptomes associated
with each chromosome, the theory predicts that resistancespecific karyotypic alterations are associated with numerous
unselected new phenotypes such as multidrug resistance.
The necessary karyotypic alterations are generated by
cancer-specific aneuploidy autocatalytically, because aneuploidy destabilizes the karyotype by misbalancing teams of
proteins that synthesize, repair and segregate chromosomes.
The rates of these alterations are proportional to the degree
of aneuploidy (Duesberg et al., 2004, 1998; Fabarius et al.,
2003). In the presence of cytotoxic drugs resistance-specific
alterations are selected from the resulting variants by classical Darwinian mechanisms (Duesberg et al., 2000, 2001; Li
et al., 2005). The resulting level of resistance would then be
proportional to the numbers of resistance-specific chromosomal alterations (Li et al., 2005) or “tumor heterogeneity” as
proposed by Alaoui-Jamali et al. (2004). Thus, aneuploidy is
the primary cause of cancer-specific drug resistance.
The theory also predicts enhanced gene mutation rates
owing to faulty DNA repair or synthesis by unbalanced teams
of enzymes. But, in contrast to mutations generated by mutator genes which are suicidal, negative mutations generated by
chromosomal imbalances would be functionally reversible
by compensatory chromosomal alterations (Duesberg et al.,
2004).
In the following paragraphs we show, by way of examples,
how the karyotypic theory can explain the four landmarks of
cancer-specific drug resistance that genetic and epigenetic
theories have failed to explain.
3
alterations. To test this prediction of the chromosomal theory,
we have recently compared the chromosomes of puromycinresistant clones derived from three human cancer lines, HT
29, SW 480 and MDA 231, with the chromosomes of drug
sensitive precursors (Li et al., 2005). The protein synthesis
inhibitor puromycin was used because it is neither mutagenic nor clastogenic and is effective in non-dividing cells.
The karyotypes of these cells were obtained from metaphase
chromosomes hybridized in situ with fluorescent color-coded
chromosome-specific DNA probes.
In view of the inherent chromosomal instability of aneuploid karyotypes, the karyotype of each resistant clone was
compared to 3–5 independent clones of the drug sensitive
parental cell lines. It can be seen in Table 1 that each resistant clone had lost between three and four parental clonal
aneusomies. There were no gains of parental chromosomes
above the low rates also observed in the parental clones.
By contrast, each resistant clone of the three cancer cell
lines had gained between one and three new, clonal hybrid
(marker) chromosomes, compared to the parental line. This is
exceptionally specific, because new, unselected marker chromosomes rarely ever become stable stemlines in cultures of
cancer cells (Li et al., 2005). Since none of 11 clones of the
parental cells grown in the absence of selective drugs acquired
new, clonal marker chromosomes, it was concluded that these
new clonal marker chromosomes are resistance-specific (Li
et al., 2005).
In addition, we have found that artificial transfer of chromosome 7 – but not of chromosome 13 – renders the human
DLD colon cancer line puromycin-resistant (Upender et al.,
2004) (unpublished).
Thus, clonal karyotype alterations coincide with drug
resistance. These new results confirm and extend several
studies in the last 50 years, which have shown correlations between gains and losses of chromosomes and
cancer-specific drug resistance (see above, Landmark 2.1),
(McDonald et al., 2005; Richardson and Kaye, 2005;
Schoenlein, 1993; Yasui et al., 2004).
A comparison of the structures of the puromycin
resistance-specific chromosomal alterations of the four different human colon cancer lines indicates, that most but not
all of the resistance-specific chromosomal alterations were
unique for each cancer cell (not shown here) (Li et al., 2005).
The same is true for the resistance-specific chromosomal
alterations described by others. The diversity of resistance-
4. Experimental evidence for the karyotypic theory
of cancer-specific drug resistance
4.1. Clonal karyotypic alterations
4.1.1. Diversity of resistance-specific karyotypes
The karyotypic theory postulates that drug resistant cancer
cells differ from sensitive precursors in new, clonal karyotype
Table 1
Numbers of clonal, chromosomal alterations acquired by human cancer cells together with resistance against puromycin (data from Li et al. (2005))
HT 29 clones
Chromosome losses
Chromosome gains
New marker
chromosomes
a
SW 480 clones
MDA clones
Averages per clone
Selected for
resistance
Parental, no
selection
Selected for
resistance
Parental, no
selection
Selected for
resistance
Parental, no
selection
Selected for
resistance
Parental, no
selection
4, 4
1, 0
1, 1
1, 0, 0
0, 0, 0
0, 0, 0
5, 3
0, 0
3, 3
1, 0, 0
1, 0, 0
0, 0, 0
2, ?a
0, ?a
2, 3
2, 0, 0, 0, 0
0, 0, 0, 0, 0
0, 0, 0, 0, 0
3.6
0.25
2
0.27
0.14
0
Uncertain because of near-tetraploidization of this clone.
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specific chromosome alterations, even against the same drug,
can thus be interpreted as a non-specific consequence of a
common, unknown cause, as for example a mutation. Accordingly, gains of chromosomes were interpreted in recent
studies as activations of resistance genes or drug export
genes and losses were interpreted as in-activations of drug
accepting or metabolizing genes (McDonald et al., 2005;
Richardson and Kaye, 2005; Schoenlein, 1993; Yasui et al.,
2004). The common flaws of these gene-based interpretations are, however, that (i) they fail to explain the restriction
of drug resistance to cancer cells, and that (ii) there is no
evidence that the respective resistance genes can transform
drug sensitive to drug-resistant cells without altering their
karyotypes.
We propose that the diversity of the resistance-specific
chromosomal alterations reflects a necessary condition of
cancer-specific drug resistance. In view of the fact that virtually all cancers have unique karyotypes (Heim and Mitelman,
1995), it can be argued that resistance-specific chromosomal
alterations must be compatible with the unique karyotypes
of the drug-sensitive progenitor cell. Likewise chromosomal alterations cause species-specific diseases, e.g. Down
syndrome, whereas mutations of essential genes cause the
same diseases in different species with different karyotypes.
This view predicts (i) that the degree of resistance is proportional to the number of resistance-specific chromosomal
alterations, and (ii) that resistance depends on complex,
resistance-specific transcriptomes, rather than on genetic or
epigenetic alteration of specific genes. Evidence confirming
these predictions is described below.
4.1.2. Resistance proportional to the number of
resistance-specific chromosomes
The karyotypic theory predicts that the progressive
increase of cancer-specific drug resistance depends on the
number of resistance-specific chromosomes. The classical
example of a progressive increase in cancer-specific drug
resistance is the rise of methotrexate resistance in cancer cells
in the presence of this drug (Law, 1952).
In vitro analyses of progressive drug resistance in the
1980s have revealed that the level of methotrexate resistance
of some cancer cells is directly proportional to the number of
extra- or intra chromosomal amplicons made of specific chromosomal segments (Schimke, 1984; Schoenlein, 1993; Tlsty,
1990). The extra-chromosomal amplicons are cytogenetically termed “minute” chromosomes (Heim and Mitelman,
1995). Such amplicons may be as small as one megabase or
even smaller (Schoenlein, 1993). These resistance-specific
amplicons are analogous to the bacterial drug resistancespecific plasmids. Much as in the case of cancer cells
with resistance-specific amplicons the dosage of resistancespecific plasmids is variable and determines the degree of
bacterial drug resistance. Moreover, biochemical studies have
shown that cancer-specific amplicons originate from unrepaired DNA breaks (Singer et al., 2000). Since DNA breaks
are common in cancer cells, owing to unbalanced teams of
DNA repair and synthesis enzymes, the karyotypic theory
also explains why resistance via amplicons is cancer-specific.
As shown in Table 1 and elsewhere, drug resistance of
cancer cells is also proportional to the number of specific
intact or marker chromosomes (Li et al., 2005; McDonald
et al., 2005; Yasui et al., 2004). This view is supported by
the recent discovery that specific chromosomal alterations
render yeast resistant against antifungal agents. As in cancer cells, in yeast “increases and decreases in drug resistance
were strongly associated with gain and loss of [an] isochromosome” (Selmecki et al., 2006).
4.1.3. Resistance-specific karyotypic alterations encode
complex transcriptomes
The expression of the thousands of genes of the chromosomes altered in cancer cells is proportional to the degree
of aneuploidy (Aggarwal et al., 2005; Duesberg et al., 2005;
Pollack et al., 2002; Tsafrir et al., 2006). This association
also appears to be true for the specific chromosomal alterations of drug-resistant cancer cells (Gyorffy et al., 2006;
Van ’t Veer et al., 2002; Yasui et al., 2004). For example,
a recent study comparing melanoma cells that had acquired
resistance to cisplatin, etoposide, fotemustine and vindesine,
“found that 21–25% of the [30,000 tested] genes were differentially expressed in each drug-resistant cell line relative
to the parental line” (Gyorffy et al., 2006). Moreover, a clinical study has found that breast cancers with the same gene
expression profiles have the same treatment responses (Van
’t Veer et al., 2002). Thus clinical and in vitro-studies both
show that resistance-specific karyotypic alterations generate
complex new transcriptomes.
In sum, cancer-specific drug resistance correlates with the
numbers, structures and expressions of clonal chromosomal
alterations and vary with the distinct karyotypes of cancer
cells.
4.2. High rates of karyotypic alterations in cancer cells
explain high rates of drug resistance
The karyotypes of human diploid cells in clonal cultures
typically remain identical for many cell generations because
the chromosomes of diploid human cells are stable (Duesberg
et al., 2005). By contrast, the karyotypes of clonal cultures of
aneuploid cancer cells include both clonal and diverse nonclonal elements. The percentage of non-clonal karyotypes is
proportional to the degree of aneuploidy (Duesberg et al.,
2004, 1998; Fabarius et al., 2003). For example, in clonal
cultures of the aneuploid human colon and breast cancer cell
lines HT 29, SW 480 and MDA 231, 66%, 65% and 35% of
the “clonal” cells have non-clonal karyotypes that differ from
that of the predominant stemline, with karyotypes changed
per cell generation being 3, 3 and 1.5 percent respectively in
these cell lines (Li et al., 2005).
These karyotypic diversities of “clonal” cultures of HT 29,
SW 480 and MDA 231 correspond to minimal rates of 1.5–3%
karyotype alteration per cell generation. Probably even higher
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rates occur, but are masked because new karyotypic variants
replace most non-clonal karyotypes in subsequent cell generations (Li et al., 2005). In clonal cultures of highly aneuploid
Chinese hamster cells often no two cells examined in a sample
have identical karyotypes, indicating that karyotypes change
at rates of up to 100% per cell generation (Fabarius et al.,
2003).
Thus, the rates of karyotype alteration per cell generation
in highly aneuploid human cancer cells are about 10−2 per
cell generation or even higher, and hence sufficient to explain
the highest observed rates for cancer-specific drug resistance
(10−3 , see above) (Li et al., 2005).
4.3. Multidrug resistance explained by unselected
transcriptomes of chromosomes selected for resistance
against specific drugs
The karyotypic theory predicts that drug resistance is
achieved by new transcriptomes, generated by the deletion
or amplification of chromosomes. Since chromosomes contain more transcriptomes than are necessary for resistance
against one specific drug, selection for chromosome alterations that confer resistance against one specific drug will
inevitably generate new unselected ‘hitchhiker’ functions, as
for example multidrug resistance, altered cell morphology
and malignancy (see above at Section 2.3). Accordingly, gene
expression profiles of drug resistant cells differ from those of
parental drug sensitive cells in the over- or underexpression
of hundreds to thousands of genes (Gyorffy et al., 2006; Van
’t Veer et al., 2002; Yasui et al., 2004).
4.4. Intrinsic drug resistance explained by unselected
transcriptomes of karyotypes selected for oncogenic
functions
The karyotypic theory also explains intrinsic cancerspecific drug resistance by transcriptomes that are accidentally associated with karyotypes selected for oncogenic
functions (Duesberg et al., 2005).
5. Conclusions
The karyotypic theory attributes cancer-specific drug
resistance to cancer-specific aneuploidy, which generates
resistance-specific karyotypic alterations autocatalytically.
The inherent instability of aneuploidy thus explains the
enormous adaptability of cancers against cytotoxic drugs,
and dims hopes for gene-specific therapies (Alaoui-Jamali
et al., 2004; Chabner and Roberts, 2005). Resistancespecific karyotypic alterations also explain the widely known
“complexity and heterogeneity” of cancer-specific drug resistance (Alaoui-Jamali et al., 2004), e.g. multidrug resistance,
“multi-factorial” mechanisms (O’Driscoll and Clynes, 2006),
“multiple interacting factors” (Roberti et al., 2006) and the
complex “gene expression program that confers drug resis-
5
tance through multiple pathways” (Richardson and Kaye,
2005). See also the second part of this discussion on drug
resistance mechanisms in this issue (Fojo, 2007). According to our theory the diversity of karyotypic alterations with
resistance against the same drug that are found in different
cancers mirrors the diversity of cancer-specific karyotypes.
In response to these conclusions it is now often argued
that resistance of chronic myeloid leukemia against imatinib (trade named, Gleevec) reflects a secondary mutation
of a leukemia-specific, primary mutation of a cellular kinase
(Lodish et al., 2004; Varmus, 2006; Fojo, 2007) (see Box 1).
Therefore, we have adduced here new evidence that even
imatinib resistance of chronic myeloid leukemia has a chromosomal basis (Box 1).
Box 1: Does mutation or chromosomal alteration render chronic myeloid
leukemia resistant against imatinib?
It is by now a textbook story that a rearrangement of the Abl kinase causes chronic
myeloid leukemia (CML) and that “an inhibitor
of Abl kinase”, imatinib (Gleevec® ), achieves
complete remissions of CML (Lodish et al.,
2004; Varmus, 2006). Soon after the introduction of imatinib, however, both intrinsic and
acquired resistance have been observed in
the “accelerated phase” and the “blast crisis”
of the disease, but not in the chronic phase
(Chabner and Roberts, 2005; Lahaye et al., 2005;
McCormick, 2001). Resistance against imatinib
therapy during the accelerated phase and blast
crisis is now widely attributed to mutations
of the Abl kinase (Chabner and Roberts, 2005;
Krause and Van Etten, 2005; McCormick, 2001).
Yet no mutated Abl kinase is found in over
half of imatinib-resistant CML cases (Lahaye
et al., 2005; McCormick, 2001). Moreover, even
in cases where mutated Abl kinase is found,
expression of the Abl gene is so low that it is
only detectable by in vitro amplification with
the polymerase chain reaction (Lahaye et al.,
2005; Marcelle et al., 1989). Instead, “clonal”
karyotype alterations are found in resistant
cases, which encode new, possibly resistancespecific transcriptomes (Lahaye et al., 2005;
Loriaux and Deininger, 2004).
The mutation theory cannot answer the following questions:
(1) Why is resistance observed only in the
accelerated phase and blast crisis, but not
in the chronic phase?
(2) Why does intrinsic resistance develop in the
absence of imatinib, i.e. prior to treatment?
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(3) How do CML cases with un-mutated Abl
resist imatinib?
(4) Why are there “clonal” karyotype alterations in imatinib resistant cells compared
to sensitive cells?
(5) Based on the classic model for the relation
between gene expression and phenotypes,
the lysogenic phage Lambda, unexpressed
genes have no phenotype (Ptashne, 2004).
Likewise resistance of human cells against
methotrexate is proportional to the dosage
of equally expressed, resistance-specific
amplicons (see Section 4.1.2). Therefore it
is hard to understand how the low amounts
of mutated Abl mRNA, which are only
detectable by the polymerase chain reaction, could suffice to protect cancer cells
against 400–600 mg imatinib per day per
patient (Lahaye et al., 2005) – or cause
leukemia for that matter.
The chromosomal theory, however, holds that
imatinib resistance is due to chromosomal
alterations. Indeed, resistant cells of the accelerated phase and blast crisis of CML are 58%
aneuploid based on conventional cytogenetic
analysis (Lahaye et al., 2005) and 100% (39/39)
aneuploid based on comparative hybridization
with gene arrays (Hosoya et al., 2006) and also
carry new, “clonal” chromosome alterations
(Lahaye et al., 2005; Loriaux and Deininger,
2004). Accordingly the five unanswered questions of the mutation theory can be answered:
(1) Resistance is not observed in diploid cells
of the chronic phase of CML, because resistance depends on aneuploidy.
(2) Intrinsic resistance derives from unselected
transcriptomes associated with chromosome alterations that are specific for the
accelerated phase and blast crisis of CML.
(3) CML cases without (and with) Abl mutations are imatinib-resistant, because resistance is chromosomal rather than dependent on kinase mutations.
(4) The new clonal karyotypes of imatinib
resistant cases are resistance-specific aneusomies.
(5) The chromosomal theory proposes that
the drug-resistance phenotype of aneuploid
CML cells is generated by the abnormal
expression of thousands of genes, rather
than by mutation of one inactive kinase
gene – if it is present.
The chromosomal theory implies that imatinib
is chemotherapeutic, because of cytotoxicity
(Krause and Van Etten, 2005; Lahaye et al., 2005)
that is independent of the Abl kinase. This toxicity, like that of conventional chemotherapeutic
drugs, is neutralized by specific karyotypic
alterations. This view is consistent with the toxicity of imatinib against many different cancers
(Krause and Van Etten, 2005) and normal cells
(Fabarius et al., 2005).
The karyotypic theory also resolves Foulds’ paradox of
the “differential utilization of the genome” by cancer cells: It
is aneuploidy-catalyzed, cancer-specific karyotypic variation
that restricts high rates and wide ranges of drug resistance to
cancer cells and that explains, why cancer genes do not confer
drug resistance to normal diploid cells in the absence of karyotypic alterations. Since karyotypic variation is incompatible
with sexual reproduction and normal development (Vogel and
Motulsky, 1986), the karyotypic theory also explains why,
despite numerous selective advantages, drug resistance is not
heritable.
Acknowledgments
We thank George Miklos (Sydney, Australia) and Thomas
Ried (National Cancer Institute, Bethesda, MD) for critical
reviews of the manuscript. We are grateful for support and
encouragement to the Foundation for Advancement in Cancer
Therapy (New York), the Abraham J. and Phyllis Katz Foundation (New York), Dr. Herbert Bernheim (philanthropist,
Miami FL), Robert Leppo (philanthropist, San Francisco),
other private sources, Thomas Ried and the Forschungsfonds
der Fakultaet fuer Klinische Medizin Mannheim, Germany.
RS is grateful to NASA for support by grant NSCOR04-00140017.
References
Aggarwal, A., Leong, S.H., Lee, C., Kon, O.L., Tan, P., 2005. Wavelet transformations of tumor expression profiles reveals a pervasive genome-wide
imprinting of aneuploidy on the cancer transcriptome. Cancer Res. 65,
186–194.
Alaoui-Jamali, M.A., Dupre, I., Qiang, H., 2004. Prediction of drug sensitivity and drug resistance in cancer by transcriptional and proteomic
profiling. Drug Resist. Updates 7, 245–255.
Allen, J.D., Brinkhuis, R.F., van Deemter, L., Wijnholds, J., Schinkel,
A.H., 2000. Extensive contribution of the multidrug transporters Pglycoprotein and Mrp1 to basal drug resistance. Cancer Res. 60,
5761–5766.
Beadle, G.W., Tatum, E.L., 1941. Genetic control of biochemical reactions
in neurospora. Proc. Natl. Acad. Sci. U.S.A. 27, 499–506.
Chabner, B.A., Roberts, T.G., 2005. Timeline: chemotherapy and the war on
cancer. Nat. Rev. Cancer 5, 65–72.
Please cite this article in press as: Duesberg, P. et al., Cancer drug resistance: The central role of the karyotype, Drug Resist. Updat. (2007),
doi:10.1016/j.drup.2007.02.003
YDRUP-396;
No. of Pages 8
ARTICLE IN PRESS
P. Duesberg et al. / Drug Resistance Updates xxx (2007) xxx–xxx
Chen, K.G., Wang, Y.C., Schaner, M.E., Francisco, B., Duran, G.E.,
Juric, D., Huff, L.M., Padilla-Nash, H., Ried, T., Fojo, T., Sikic, B.I.,
2005. Genetic and epigenetic modeling of the origins of multidrugresistant cells in a human sarcoma cell line. Cancer Res. 65, 9388–
9397.
Duesberg, P., Fabarius, A., Hehlmann, R., 2004. Aneuploidy, the primary
cause of the multilateral genomic instability of neoplastic and preneoplastic cells. IUBMB Life 56, 65–81.
Duesberg, P., Li, R., Fabarius, A., Hehlmann, R., 2005. The chromosomal
basis of cancer. Cell Oncol. 27, 293–318.
Duesberg, P., Rausch, C., Rasnick, D., Hehlmann, R., 1998. Genetic instability of cancer cells is proportional to their degree of aneuploidy. Proc.
Natl. Acad. Sci. U.S.A. 95, 13692–13697.
Duesberg, P., Stindl, R., Hehlmann, R., 2000. Explaining the high mutation
rates of cancer cells to drug and multidrug resistance by chromosome
reassortments that are catalyzed by aneuploidy. Proc. Natl. Acad. Sci.
U.S.A. 97, 14295–14300.
Duesberg, P., Stindl, R., Hehlmann, R., 2001. Origin of multidrug resistance
in cells with and without multidrug resistance genes: chromosome reassortments catalyzed by aneuploidy. Proc. Natl. Acad. Sci. U.S.A. 98,
11283–11288.
Fabarius, A., Giehl, M., Frank, O., Duesberg, P., Hochhaus, A., Hehlmann,
R., Seifarth, W., 2005. Induction of centrosome and chromosome aberrations by imatinib in vitro. Leukemia 19, 1573–1578.
Fabarius, A., Hehlmann, R., Duesberg, P.H., 2003. Instability of chromosome structure in cancer cells increases exponentially with degrees of
aneuploidy. Cancer Genet. Cytogenet. 143, 59–72.
Fabarius, A., Willer, A., Yerganian, G., Hehlmann, R., Duesberg, P., 2002.
Specific aneusomies in Chinese hamster cells at different stages of neoplastic transformation, initiated by nitrosomethylurea. Proc. Natl. Acad.
Sci. U.S.A. 99, 6778–6783.
Fojo, T., 2007. Multiple paths to a drug resistance phenotype: mutations,
translocations, deletions and amplification of coding genes or promoter
regions, epigenetic changes and micro RNAs. Drug Resist. Updates 10,
doi:10.1016/j.drup.2007.02.002.
Foulds, L., 1969. Neoplastic Development, vol. 2. Academic Press, London,
New York, San Francisco.
Goldie, J.H., 2001. Drug resistance in cancer: a perspective. Cancer Metastasis Rev. 20, 63–68.
Gyorffy, B., Serra, V., Materna, V., Schafer, R., Dietel, M., Schadendorf, D.,
Lage, H., 2006. Analysis of gene expression profiles in melanoma cells
with acquired resistance against antineoplastic drugs. Melanoma Res.
16, 147–155.
Hartman, J.L., Garvik, B., Hartwell, L., 2001. Principles for the buffering of
genetic variation. Science 291, 1001–1004.
Heim, S., Mitelman, F., 1995. Cancer Cytogenetics. Wiley-Liss, New York.
Heselmeyer, K., Macville, M., Schrock, E., Blegen, H., Hellstrom, A.C.,
Shah, K., Auer, G., Ried, T., 1997. Advanced-stage cervical carcinomas
are defined by a recurrent pattern of chromosomal aberrations revealing
high genetic instability and a consistent gain of chromosome arm 3q.
Genes Chromosomes Cancer 19, 233–240.
Hosoya, N., Sanada, M., Nannya, Y., Nakazaki, K., Wang, L., Hangaishi, A.,
Kurokawa, M., Chiba, S., Ogawa, S., 2006. Genomewide screening of
DNA copy number changes in chronic myelogenous leukemia with the
use of high-resolution array-based comparative genomic hybridization.
Genes Chromosomes Cancer 45, 482–494.
Kacser, H., Burns, J.A., 1981. The molecular basis of dominance. Genetics
97, 639–666.
Kotchetkov, R., Driever, P.H., Cinatl, J., Michaelis, M., Karaskova, J.,
Blaheta, R., Squire, J.A., Von Deimling, A., Moog, J., Cinatl Jr., J.,
2005. Increased malignant behavior in neuroblastoma cells with acquired
multi-drug resistance does not depend on P-gp expression. Int. J. Oncol.
27, 1029–1037.
Krause, D.S., Van Etten, R.A., 2005. Tyrosine kinases as targets for cancer
therapy. New Engl. J. Med. 353, 172–187.
Lahaye, T., Riehm, B., Berger, U., Paschka, P., Muller, M.C., Kreil, S.,
Merx, K., Schwindel, U., Schoch, C., Hehlmann, R., Hochhaus, A.,
7
2005. Response and resistance in 300 patients with BCR-ABL-positive
leukemias treated with imatinib in a single center: a 4.5-year follow-up.
Cancer 103, 1659–1669.
Law, L.W., 1952. Origin of the resistance of leukaemic cells to folic acid
antagonists. Nature 169, 628–629.
Lengauer, C., Kinzler, K.W., Vogelstein, B., 1998. Genetic instabilities in
human cancers. Nature 396, 643–649.
Lewin, B., 1997. Genes VI. Oxford University Press, Oxford, Chapter 4.
Li, R., Hehlmann, R., Sachs, R., Duesberg, P., 2005. Chromosomal alterations cause the high rates and wide ranges of drug resistance in cancer
cells. Cancer Genet. Cytogenet. 163, 44–56.
Lodish, H., Berk, A., Matsudaira, P., Kaiser, C.A., Krieger, M., Scott, M.P.,
Zipursky, S.L., Darnell, J., 2004. Molecular Cell Biology. W.H. Freeman,
New York, Basingstoke, UK.
Loriaux, M., Deininger, M., 2004. Clonal cytogenetic abnormalities
in Philadelphia chromosome negative cells in chronic myeloid
leukemia patients treated with imatinib. Leuk. Lymphoma 45, 2197–
2203.
Marcelle, C., Gale, R.P., Prokocimer, M., Berrebi, A., Merle-Beral, H.,
Canaani, E., 1989. Analysis of BCR-ABL mRNA in chronic myelogenous leukemia patients and identification of a new BCR-related sequence
in human DNA. Genes Chromosomes Cancer 1, 172–179.
Marx, J., 2002. Debate surges over the origins of genomic defects in cancer.
Science 297, 544–546.
McCormick, F., 2001. New-age drug meets resistance. Nature 412, 281–282.
McDonald, S.L., Stevenson, D.A., Moir, S.E., Hutcheon, A.W., Haites, N.E.,
Heys, S.D., Schofield, A.C., 2005. Genomic changes identified by comparative genomic hybridisation in docetaxel-resistant breast cancer cell
lines. Eur. J. Cancer 41, 1086–1094.
Michor, F., Nowak, M.A., Iwasa, Y., 2006. Evolution of resistance to cancer
therapy. Curr. Pharm. Des. 12, 261–271.
Morgillo, F., Lee, H.Y., 2005. Resistance to epidermal growth factor
receptor-targeted therapy. Drug Resist. Updates 8, 298–310.
O’Driscoll, L., Clynes, M., 2006. Biomarkers and multiple drug resistance
in breast cancer. Curr. Cancer Drug Targets 6, 365–384.
Pollack, J.R., Sorlie, T., Perou, C.M., Rees, C.A., Jeffrey, S.S., Lonning, P.E.,
Tibshirani, R., Botstein, D., Borresen-Dale, A.L., Brown, P.O., 2002.
Microarray analysis reveals a major direct role of DNA copy number
alteration in the transcriptional program of human breast tumors. Proc.
Natl. Acad. Sci. U.S.A. 99, 12963–12968.
Ptashne, M., 2004. A Genetic Switch: Phage Lambda Revisited. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY.
Richardson, A., Kaye, S.B., 2005. Drug resistance in ovarian cancer: the
emerging importance of gene transcription and spatio-temporal regulation of resistance. Drug Resist. Updates 8, 311–321.
Roberti, A., La Sala, D., Cinti, C., 2006. Multiple genetic and epigenetic
interacting mechanisms contribute to clonally selection of drug-resistant
tumors: current views and new therapeutic prospective. J. Cell. Physiol.
207, 571–581.
Ross, D.T., Scherf, U., Eisen, M.B., Perou, C.M., Rees, C., Spellman, P.,
Iyer, V., Jeffrey, S.S., Van de Rijn, M., Waltham, M., Pergamenschikov,
A., Lee, J.C., Lashkari, D., Shalon, D., Myers, T.G., Weinstein, J.N.,
Botstein, D., Brown, P.O., 2000. Systematic variation in gene expression
patterns in human cancer cell lines. Nat. Genet. 24, 227–235.
Schimke, R.T., 1984. Gene amplification, drug resistance, and cancer. Cancer
Res. 44, 1735–1742.
Schoenlein, P.V., 1993. Molecular cytogenetics of multiple drug resistance.
Cytotechnology 12, 63–89.
Selmecki, A., Forche, A., Berman, J., 2006. Aneuploidy and isochromosome
formation in drug-resistant Candida albicans. Science 313, 367–370.
Singer, M.J., Mesner, L.D., Friedman, C.L., Trask, B.J., Hamlin, J.L., 2000.
Amplification of the human dihydrofolate reductase gene via double
minutes is initiated by chromosome breaks. Proc. Natl. Acad. Sci. U.S.A.
97, 7921–7926.
Terzi, M., 1974. Chromosomal variation and the origin of drug-resistant
mutants in mammalian cell lines. Proc. Natl. Acad. Sci. U.S.A. 71,
5027–5031.
Please cite this article in press as: Duesberg, P. et al., Cancer drug resistance: The central role of the karyotype, Drug Resist. Updat. (2007),
doi:10.1016/j.drup.2007.02.003
YDRUP-396;
8
No. of Pages 8
ARTICLE IN PRESS
P. Duesberg et al. / Drug Resistance Updates xxx (2007) xxx–xxx
Tlsty, T.D., 1990. Normal diploid human and rodent cells lack a detectable
frequency of gene amplification. Proc. Natl. Acad. Sci. U.S.A. 87,
3132–3136.
Tomlinson, I.P., Novelli, M.R., Bodmer, W.F., 1996. The mutation rate and
cancer. Proc. Natl. Acad. Sci. U.S.A. 93, 14800–14803.
Tsafrir, D., Bacolod, M., Selvanayagam, Z., Tsafrir, I., Shia, J., Zeng, Z.,
Liu, H., Krier, C., Stengel, R.F., Barany, F., Gerald, W.L., Paty, P.B.,
Domany, E., Notterman, D.A., 2006. Relationship of gene expression
and chromosomal abnormalities in colorectal cancer. Cancer Res. 66,
2129–2137.
Upender, M.B., Habermann, J.K., McShane, L.M., Korn, E.L., Barrett, J.C.,
Difilippantonio, M.J., Ried, T., 2004. Chromosome transfer induced
aneuploidy results in complex dysregulation of the cellular transcriptome in immortalized and cancer cells. Cancer Res. 64, 6941–
6949.
Van ’t Veer, L.J., Dai, H., van de Vijver, M.J., He, Y.D., Hart, A.A., Mao, M.,
Peterse, H.L., van der Kooy, K., Marton, M.J., Witteveen, A.T., Schreiber,
G.J., Kerkhoven, R.M., Roberts, C., Linsley, P.S., Bernards, R., Friend,
S.H., 2002. Gene expression profiling predicts clinical outcome of breast
cancer. Nature 415, 530–536.
Varmus, H., 2006. The new era in cancer research. Science 312, 1162–1165.
Vogel, F., Motulsky, A.G., 1986. Human Genetics: Problems and
Approaches. Springer Verlag, Berlin, Heidelberg, New York, Tokyo.
Vogt, M., 1959. A study of the relationship between karyotype and phenotype
in cloned lines of strain HeLa. Genetics 44, 1257–1270.
Wang, T.L., Rago, C., Silliman, N., Ptak, J., Markowitz, S., Willson, J.K.,
Parmigiani, G., Kinzler, K.W., Vogelstein, B., Velculescu, V.E., 2002.
Prevalence of somatic alterations in the colorectal cancer cell genome.
Proc. Natl. Acad. Sci. U.S.A. 99, 3076–3080.
Wright, J.A., Smith, H.S., Watt, F.M., Hancock, M.C., Hudson, D.L., Stark,
G.R., 1990. DNA amplification is rare in normal human cells. Proc. Natl.
Acad. Sci. U.S.A. 87, 1791–1795.
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.,
2004. Alteration in copy numbers of genes as a mechanism for acquired
drug resistance. Cancer Res. 64, 1403–1410.
Please cite this article in press as: Duesberg, P. et al., Cancer drug resistance: The central role of the karyotype, Drug Resist. Updat. (2007),
doi:10.1016/j.drup.2007.02.003