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YDRUP-396; No. of Pages 8 ARTICLE IN PRESS 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 YDRUP-396; 2 No. of Pages 8 ARTICLE IN PRESS P. Duesberg et al. / Drug Resistance Updates xxx (2007) xxx–xxx 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- 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; ARTICLE IN PRESS No. of Pages 8 P. Duesberg et al. / Drug Resistance Updates xxx (2007) xxx–xxx 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. 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; 4 No. of Pages 8 ARTICLE IN PRESS P. Duesberg et al. / Drug Resistance Updates xxx (2007) xxx–xxx 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 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 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? 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; 6 No. of Pages 8 ARTICLE IN PRESS P. Duesberg et al. / Drug Resistance Updates xxx (2007) xxx–xxx (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. 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