Detection of centrosome aberrations in disease-unrelated cells from patients with tumor treated with tyrosine kinase inhibitors.

OBJECTIVES: Tyrosine kinase inhibitors (TKIs) target various pathways associated with proliferation of aberrant clones in malignant diseases. Despite good response and acceptable tolerability, little is known concerning long-term toxicity. Furthermore, the influence of these inhibitors on disease-unrelated cells is not investigated yet. METHODS: Centrosome aberrations are hallmarks of various cancers. We sought to evaluate the effect of TKIs on centrosomes of disease-unrelated cells. We examined cells of the oral mucosa (OM) and fibroblasts of patients with chronic myeloid leukemia (CML) treated with dasatinib and bosutinib. Results were compared with data from patients with CML treated with imatinib or nilotinib and with data from patients suffering from renal and hepatocellular carcinomas (RCC/HCC) treated with sorafenib or sunitinib. Cells of healthy donors served as controls. RESULTS: OM cells (n = 12) and fibroblasts (n = 7) of patients with CML treated with dasatinib and OM cells of three patients with CML treated with bosutinib showed centrosomal alterations (mean, 14%) compared with 16 (10 OM and 6 fibroblasts) controls (mean, 3%). OM cells of five patients with CML and one patient with systemic mastocytosis treated with imatinib or nilotinib and of eight patients with RCC or HCC treated with sorafenib or sunitinib showed centrosome defects in a mean of 15%. CONCLUSIONS: Our data have shown that TKI treatment of tumor patients may influence centrosomes in disease-unrelated cells or tissues. This may be important with regard to various observed side effects.

Centrosome aberrations and G1 phase arrest after in vitro and in vivo treatment with the SRC/ABL inhibitor dasatinib.

BACKGROUND: Dasatinib is a multitargeted inhibitor of ABL, the SRC family, and other tyrosine kinases. We sought to evaluate the effects of this drug on cell proliferation, centrosomes, mitotic spindles, and cell cycle progression in vitro and in vivo. DESIGN AND METHODS: Human dermal fibroblasts, Chinese hamster cells, human osteosarcoma cells, and blood and bone marrow mononuclear cells from 32 patients with chronic myeloid leukemia, gastrointestinal stromal tumor, and systemic mastocytosis as well as from six healthy individuals were investigated. The effects of dasatinib were compared with those of the ABL inhibitors imatinib and nilotinib, the SRC inhibitor PP2, and the ABL/LYN inhibitor INNO-406. RESULTS: Dasatinib induced G(1) phase arrest in all cell lines and this was associated with a decline in cyclin D1 levels. In vitro, centrosomal aberrations, a decrease of mitotic spindles, and G(1) phase arrest were observed. In patients, centrosome alterations were found in a median of 17% (range, 10-22%) of cells with a decrease of spindles in 8/18 patients. In comparison, imatinib, nilotinib and PP2 led to centrosome aberrations without G(1) phase arrest. INNO-406 was associated with centrosome aberrations and cell cycle arrest in G(1) phase. CONCLUSIONS: Dasatinib blocks the G(1)/S transition and inhibits cell growth. It induces centrosomal aberrations and a decrease of mitotic spindles. The effects suggest an involvement of SRC and ABL inhibition.

Specific clones of spontaneously evolving karyotypes generate individuality of cancers.

Several researchers, including us, have recently proposed that specific karyotypes, rather than specific mutations, generate the "biochemical individuality" of cancers, defined by individual growth rates, metabolisms, drug-resistances, metastases and cell morphologies. According to our theory, independent karyotypic evolutions generate cancers, much like new phylogenetic species. To allow such evolutions in the lifetime of an organism, the normal karyotype must be destabilized, but not the genes. The karyotype is destabilized by aneuploidy, because aneuploidy unbalances conserved teams of proteins that segregate, synthesize and repair chromosomes. And aneuploidy is induced either by carcinogens or spontaneously. Here, we tested this theory using a new system that virtually excludes spontaneous mutation. In this sytem, 50% of normal human muscle cells became aneuploid and 5 per 10(6) formed foci of transformed Mu6 cells - only 2 months after transfection with 6 virus-activated cellular genes. Analyses of 10 foci revealed: (1) clonal karyotypes, consisting of one or more stemlines of spontaneously evolving aneuploidies and some non-clonal aneuploidies, and (2) individual phenotypes, such as cell morphologies, growth rates and intrinsic resistance to cytosine arabinoside, shared by 5 foci with a common stemline. Due to the short preneoplastic latencies of Mu6 cells several non-clonal precursors of focus-specific, aneuploid karyotypes were detectable before focus formation. Chemical carcinogens were also found to induce tumors with clonally evolving stemlines in Chinese hamsters. We conclude that specific clones of spontaneously evolving karyotypes, rather than specific mutations, generate the individuality of cancers. This answers the age-old question, why even cancers of the same kind do not have consistent karyotypes.

Is cancer progression caused by gradual or simultaneous acquisitions of new chromosomes?

BACKGROUND: Foulds defined, "Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters" (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained "genetic instability" generates "variant sublines (with) changes in chromosome number" and that "clonal" progressions arise by "stepwise selection of more aggressive sublines". The literature, however, contains many examples of "immediate" selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates. RESULTS: In view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates "immediate" progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of "immediate" progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process. CONCLUSIONS: We conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the "late" clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression, confirming classical predictions based on DNA content.

Speciation Theory of Carcinogenesis Explains Karyotypic Individuality and Long Latencies of Cancers.

It has been known for over 100 years that cancers have individual karyotypes and arise only years to decades after initiating carcinogens. However, there is still no coherent theory to explain these definitive characteristics of cancer. The prevailing mutation theory holds that cancers are late because the primary cell must accumulate 3⁻8 causative mutations to become carcinogenic and that mutations, which induce chromosomal instability (CIN), generate the individual karyotypes of cancers. However, since there is still no proven set of mutations that transforms a normal to a cancer cell, we have recently advanced the theory that carcinogenesis is a form of speciation. This theory predicts carcinogens initiate cancer by inducing aneuploidy, which automatically unbalances thousands of genes and thus catalyzes chain-reactions of progressive aneuploidizations. Over time, these aneuploidizations have two endpoints, either non-viable karyotypes or very rarely karyotypes of new autonomous and immortal cancers. Cancer karyotypes are immortalized despite destabilizing congenital aneuploidy by clonal selections for autonomy-similar to those of conventional species. This theory predicts that the very low probability of converting the karyotype of a normal cell to that of a new autonomous cancer species by random aneuploidizations is the reason for the karyotypic individuality of new cancers and for the long latencies from carcinogens to cancers. In testing this theory, we observed: (1) Addition of mutagenic and non-mutagenic carcinogens to normal human and rat cells generated progressive aneuploidizations months before neoplastic transformation. (2) Sub-cloning of a neoplastic rat clone revealed heritable individual karyotypes, rather than the non-heritable karyotypes predicted by the CIN theory. (3) Analyses of neoplastic and preneoplastic karyotypes unexpectedly identified karyotypes with sets of 3⁻11 new marker chromosomes without detectable intermediates, consistent with single-step origins. We conclude that the speciation theory explains logically the long latencies from carcinogen exposure and the individuality of cancers. In addition, the theory supports the single-step origins of cancers, because karyotypic autonomy is all-or-nothing. Accordingly, we propose that preneoplastic aneuploidy and clonal neoplastic karyotypes provide more reliable therapeutic indications than current analyses of thousands of mutations.

Aneuploidy and cancer: from correlation to causation.

Conventional genetic theories have failed to explain why cancer (1) is not found in newborns and thus not heritable; (2) develops only years to decades after 'initiation' by carcinogens; (3) is caused by non-mutagenic carcinogens; (4) is chromosomally and phenotypically 'unstable'; (5) carries cancer-specific aneuploidies; (6) evolves polygenic phenotypes; (7) nonselective phenotypes such as multidrug resistance, metastasis or affinity for non-native sites and 'immortality' that is not necessary for tumorigenesis; (8) contains no carcinogenic mutations. We propose instead that cancer is a chromosomal disease: Accordingly, carcinogens initiate chromosomal evolutions via unspecific aneuploidies. By unbalancing thousands of genes aneuploidy corrupts teams of proteins that segregate, synthesize and repair chromosomes. Aneuploidy is thus a steady source of karyotypic-phenotypic variations from which, in classical Darwinian terms, selection of cancer-specific aneuploidies encourages the evolution and subsequent malignant 'progressions' of cancer cells. The rates of these variations are proportional to the degrees of aneuploidy, and can exceed conventional mutation by 4-7 orders of magnitude. This makes cancer cells new cell 'species' with distinct, but unstable karyotypes, rather than mutant cells. The cancer-specific aneuploidies generate complex, malignant phenotypes, through the abnormal dosages of the thousands of genes, just as trisomy 21 generates Down syndrome. Thus cancer is a chromosomal rather than a genetic disease. The chromosomal theory explains (1) nonheritability of cancer, because aneuploidy is not heritable; (2) long 'neoplastic latencies' by the low probability of evolving competitive new species; (3) nonselective phenotypes via genes hitchhiking on selective chromosomes, and (4) 'immortality', because chromosomal variations neutralize negative mutations and adapt to inhibitory conditions much faster than conventional mutation. Based on this article a similar one, entitled 'The chromosomal basis of cancer', has since been published by us in Cellular Oncology 2005;27:293-318.

Inherent variability of cancer-specific aneuploidy generates metastases.

BACKGROUND: The genetic basis of metastasis is still unclear because metastases carry individual karyotypes and phenotypes, rather than consistent mutations, and are rare compared to conventional mutation. There is however correlative evidence that metastasis depends on cancer-specific aneuploidy, and that metastases are karyotypically related to parental cancers. Accordingly we propose that metastasis is a speciation event. This theory holds that cancer-specific aneuploidy varies the clonal karyotypes of cancers automatically by unbalancing thousands of genes, and that rare variants form new autonomous subspecies with metastatic or other non-parental phenotypes like drug-resistance - similar to conventional subspeciation. RESULTS: To test this theory, we analyzed the karyotypic and morphological relationships between seven cancers and corresponding metastases. We found (1) that the cellular phenotypes of metastases were closely related to those of parental cancers, (2) that metastases shared 29 to 96% of their clonal karyotypic elements or aneusomies with the clonal karyotypes of parental cancers and (3) that, unexpectedly, the karyotypic complexity of metastases was very similar to that of the parental cancer. This suggests that metastases derive cancer-specific autonomy by conserving the overall complexity of the parental karyotype. We deduced from these results that cancers cause metastases by karyotypic variations and selection for rare metastatic subspecies. Further we asked whether metastases with multiple metastasis-specific aneusomies are assembled in one or multiple, sequential steps. Since (1) no stable karyotypic intermediates of metastases were observed in cancers here and previously by others, and (2) the karyotypic complexities of cancers are conserved in metastases, we concluded that metastases are generated from cancers in one step - like subspecies in conventional speciation. CONCLUSIONS: We conclude that the risk of cancers to metastasize is proportional to the degree of cancer-specific aneuploidy, because aneuploidy catalyzes the generation of subspecies, including metastases, at aneuploidy-dependent rates. Since speciation by random chromosomal rearrangements and selection is unpredictable, the theory that metastases are karyotypic subspecies of cancers also explains Foulds' rules, which hold that the origins of metastases are "abrupt" and that their phenotypes are "unpredictable."

The chromosomal basis of cancer.

Conventional genetic theories have failed to explain why cancer (1) is not heritable and thus extremely rare in newborns, (2) is caused by non-mutagenic carcinogens, (3) develops only years to decades after initiation by carcinogens, (4) follows pre-neoplastic aneuploidy, (5) is aneuploid, (6) is chromosomally and phenotypically "unstable", (7) carries specific aneusomies, (8) generates much more complex phenotypes than conventional mutation such as multidrug resistance, (9) generates nonselective phenotypes such as metastasis (no benefit at native site) and "immortality" (not necessary for tumorigenesis), and (10) does not contain carcinogenic mutations. We propose, instead, that cancer is a chromosomal disease. Accordingly carcinogenesis is initiated by random aneuploidies, which are induced by carcinogens or spontaneously. Since aneuploidy unbalances 1000s of genes, it corrupts teams of proteins that segregate, synthesize and repair chromosomes. Aneuploidy is therefore a steady source of chromosomal variations from which, in classical Darwinian terms, selection encourages the evolution and malignant progression of cancer cells. The rates of specific chromosomal variations can exceed conventional mutations by 4-11 orders of magnitude, depending on the degrees of aneuploidy. Based on their chromosomal constitution cancer cells are new cell "species" with specific aneusomies, but unstable karyotypes. The cancer-specific aneusomies generate complex, malignant phenotypes through the abnormal dosages of 1000s of genes, just as trisomy 21 generates Down syndrome. In sum, cancer is caused by chromosomal disorganization, which increases karyotypic entropy. Thus, cancer is a chromosomal rather than a genetic disease. The chromosomal theory explains (1) non-heritable cancer because aneuploidy is not heritable, (2) non-mutagenic carcinogens as aneuploidogens, (3) long neoplastic latencies by the low probability of evolving new species, (4) nonselective phenotypes via genes hitchhiking with selective chromosomes, and (5) immortality because, through their cellular heterogeneity, cancers survive negative mutations and cytotoxic drugs via resistant subspecies.

Chromosomal alterations cause the high rates and wide ranges of drug resistance in cancer cells.

Conventional mutation-selection theories have failed to explain (i) how cancer cells become spontaneously resistant against cytotoxic drugs at rates of up to 10(-3) per cell generation, orders higher than gene mutation, even in cancer cells; (ii) why resistance far exceeds a challenging drug-a 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 10(-2) 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.

Multistep carcinogenesis: a chain reaction of aneuploidizations.

Carcinogenesis is a multistep process in which new, parasitic and polymorphic cancer cells evolve from a single, normal diploid cell. This normal cell is converted to a prospective cancer cell, alias "initiated", either by a carcinogen or spontaneously. The initiated cell typically does not have a new distinctive phenotype yet, but evolves spontaneously--over months to decades--to a clinical cancer. The cells of a primary cancer also evolve spontaneously towards more and more malignant phenotypes. The outstanding genotype of initiated and cancer cells is aneuploidy, an abnormal balance of chromosomes, which increases and varies in proportion with malignancy. The driving force of the spontaneous evolution of initiated and cancerous cells to ever more abnormal phenotypes is said to be their "genetic instability". However, since neither the instability of cancer phenotypes nor the characteristically slow kinetics of carcinogenesis are compatible with gene mutation, we propose here that the driving force of carcinogenesis is the inherent instability of aneuploid karyotypes. Aneuploidy renders chromosome structure and segregation error-prone, because it unbalances mitosis proteins and the many teams of enzymes that synthesize and maintain chromosomes. Thus, carcinogenesis is initiated by a random aneuploidy, which is induced either by a carcinogen or spontaneously. The resulting karyotype instability sets off a chain reaction of aneuploidizations, which generate ever more abnormal and eventually cancer-specific combinations and rearrangements of chromosomes. According to this hypothesis the many abnormal phenotypes of cancer are generated by abnormal dosages of thousands of aneuploid, but un-mutated genes.

Karyotype alteration generates the neoplastic phenotypes of SV40-infected human and rodent cells.

BACKGROUND: Despite over 50 years of research, it remains unclear how the DNA tumor viruses SV40 and Polyoma cause cancers. Prevailing theories hold that virus-coded Tumor (T)-antigens cause cancer by inactivating cellular tumor suppressor genes. But these theories don't explain four characteristics of viral carcinogenesis: (1) less than one in 10,000 infected cells become cancer cells, (2) cancers have complex individual phenotypes and transcriptomes, (3) recurrent tumors without viral DNA and proteins, (4) preneoplastic aneuploidies and immortal neoplastic clones with individual karyotypes. RESULTS: As an alternative theory we propose that viral carcinogenesis is a form of speciation, initiated by virus-induced aneuploidy. Since aneuploidy destabilizes the karyotype by unbalancing thousands of genes it catalyzes chain reactions of karyotypic and transcriptomic evolutions. Eventually rare karyotypes evolve that encode cancer-specific autonomy of growth. The low probability of forming new autonomous cancer-species by random karyotypic and transcriptomic variations predicts individual and clonal cancers. Although cancer karyotypes are congenitally aneuploid and thus variable, they are stabilized or immortalized by selections for variants with cancer-specific autonomy. Owing to these inherent variations cancer karyotypes are heterogeneous within clonal margins. To test this theory we analyzed karyotypes and phenotypes of SV40-infected human, rat and mouse cells developing into neoplastic clones. In all three systems we found (1) preneoplastic aneuploidies, (2) neoplastic clones with individual clonal but flexible karyotypes and phenotypes, which arose from less than one in 10,000 infected cells, survived over 200 generations, but were either T-antigen positive or negative, (3) spontaneous and drug-induced variations of neoplastic phenotypes correlating 1-to-1 with karyotypic variations. CONCLUSIONS: Since all 14 virus-induced neoplastic clones tested contained individual clonal karyotypes and phenotypes, we conclude that these karyotypes have generated and since maintained these neoplastic clones. Thus SV40 causes cancer indirectly, like carcinogens, by inducing aneuploidy from which new cancer-specific karyotypes evolve automatically at low rates. This theory explains the (1) low probability of carcinogenesis per virus-infected cell, (2) the individuality and clonal flexibility of cancer karyotypes, (3) recurrence of neoplasias without viral T-antigens, and (4) the individual clonal karyotypes, transcriptomes and immortality of virus-induced neoplasias - all unexplained by current viral theories.

Instability of chromosome structure in cancer cells increases exponentially with degrees of aneuploidy.

Structurally altered or marker chromosomes are the cytogenetic hallmarks of cancer cells, but their origins are still debated. Here we propose that aneuploidy, which is ubiquitous in cancer and inevitably unbalances thousands of synergistic genes, destabilizes the structure of chromosomes by catalyzing DNA breaks. Aneuploidy catalyzes such breaks by unbalancing teams of enzymes, which synthesize and maintain DNA and nucleotide pools, and even unbalancing histones via the corresponding genes. DNA breaks then initiate deletions, amplifications, and intra- and interchromosomal rearrangements. Our hypothesis predicts that the rate at which chromosomes are altered is proportional to the degree of aneuploidy: the more abnormal the number and balance of chromosomes, the higher the rate of structural alterations. To test this prediction, we have determined the rates at which clonal cultures of diploid and aneuploid Chinese hamster cells generate new, and thus nonclonal, structurally altered chromosomes per mitosis. Based on about 20 metaphases, the number of new, structurally altered chromosomes was 0 per diploid, 0-0.23 per near-diploid, 0.2-1.4 per hypotriploid, 3.25-4.8 per hypertriploid, and 0.4 per near-tetraploid cell. Thus, instability of chromosome structure increases exponentially with the deviation of ploidy from the normal diploid and tetraploid balances. The particular chromosomes engaged in aneuploidy also affected the rates of chromosome alteration, particularly at low aneuploidy indices. We conclude that aneuploidy is sufficient to cause structural instability of chromosomes. Further, we suggest that certain structurally altered chromosomes encode cancer-specific phenotypes that cannot be generated by unbalancing intact chromosomes. We also extend the evidence for aneuploidy causing numerical instability of chromosomes autocatalytically, and adduce evidence that aneuploidy can cause the many gene mutations of cancer cells that have been attributed to various mutator genes.

The chemical bases of the various AIDS epidemics: recreational drugs, anti-viral chemotherapy and malnutrition.

In 1981 a new epidemic of about two-dozen heterogeneous diseases began to strike non-randomly growing numbers of male homosexuals and mostly male intravenous drug users in the US and Europe. Assuming immunodeficiency as the common denominator the US Centers for Disease Control (CDC) termed the epidemic, AIDS, for acquired immunodeficiency syndrome. From 1981-1984 leading researchers including those from the CDC proposed that recreational drug use was the cause of AIDS, because of exact correlations and of drug-specific diseases. However, in 1984 US government researchers proposed that a virus, now termed human immunodeficiency virus (HIV), is the cause of the non-random epidemics of the US and Europe but also of a new, sexually random epidemic in Africa. The virus-AIDS hypothesis was instantly accepted, but it is burdened with numerous paradoxes, none of which could be resolved by 2003: Why is there no HIV in most AIDS patients, only antibodies against it? Why would HIV take 10 years from infection to AIDS? Why is AIDS not self-limiting via antiviral immunity? Why is there no vaccine against AIDS? Why is AIDS in the US and Europe not random like other viral epidemics? Why did AIDS not rise and then decline exponentially owing to antiviral immunity like all other viral epidemics? Why is AIDS not contagious? Why would only HIV carriers get AIDS who use either recreational or anti-HIV drugs or are subject to malnutrition? Why is the mortality of HIV-antibody-positives treated with anti-HIV drugs 7-9%, but that of all (mostly untreated) HIV-positives globally is only 1.4%? Here we propose that AIDS is a collection of chemical epidemics, caused by recreational drugs, anti-HIV drugs, and malnutrition. According to this hypothesis AIDS is not contagious, not immunogenic, not treatable by vaccines or antiviral drugs, and HIV is just a passenger virus. The hypothesis explains why AIDS epidemics strike non-randomly if caused by drugs and randomly if caused by malnutrition, why they manifest in drug- and malnutrition-specific diseases, and why they are not self-limiting via anti-viral immunity. The hypothesis predicts AIDS prevention by adequate nutrition and abstaining from drugs, and even cures by treating AIDS diseases with proven medications.

Karyotypic evolutions of cancer species in rats during the long latent periods after injection of nitrosourea.

BACKGROUND: A century of research has established that cancers arise from tissues exposed to carcinogens only after long latencies of years to decades and have individual clonal karyotypes. Since speciation from known precursors also depends on long latencies and new species also have individual karyotypes, we and others have recently proposed that carcinogenesis is a form of speciation. According to this theory karyotypic evolutions generate new cancer species from normal cells as follows: Carcinogens induce aneuploidy (Figure 1). By unbalancing thousands of genes aneuploidy automatically destabilizes the karyotype and thus catalyzes random karyotypic variations. Selections of variants with proliferative phenotypes form non-clonal hyperplasias with persistently varying karyotypes. Very rare karyotypic variations form new cancer species with individual clonal karyotypes. Despite destabilization by the resulting congenital aneuploidies, cancer karyotypes are stabilized within narrow margins of variation by clonal selections for cancer-specific autonomy. Because all non-cancerous aneuploidies are unstable, all aneusomies of prospective cancers are joined in single-steps, rather than gradually. Since this mechanism is very inefficient, it predicts long latent periods from carcinogens to cancers and individual clonal cancer karyotypes. RESULTS: Here we have tested the predicted roles of karyotypic evolutions during the time course of carcinogenesis in an established experimental system. In this system injection of nitrosourea induces in female rats non-invasive mammary hyperplasias ("tumors") after two or more months, and invasive carcinomas after six or more months. Accordingly four specific predictions were tested: (1) Invasive cancers are late and carry individual clonal karyotypes and phenotypes, (2) Persistent hyperplasias carry non-clonal karyotypes, (3) Non-clonal hyperplasias generate clonal cancers spontaneously but rarely, (4) Cancer-karyotypes arise with all individual clonal aneusomies in single-steps. All four predictions were experimentally confirmed. CONCLUSIONS: Our results along with the literature reveal a coherent karyotypic mechanism of carcinogenesis: Carcinogens induce aneuploidy. The inherent instability of aneuploidy automatically catalyzes new karyotypic variations. Aneuploid karyotypes with proliferative phenotypes form varying non-clonal hyperplasias. Rare variations form cancer species with individual clonal karyotypes, which are stabilized by clonal selection for autonomy. The low odds of this mechanism explain the long latencies of carcinogenesis, the individuality and karyotypic clonality of cancers.

Immortality of cancers: a consequence of inherent karyotypic variations and selections for autonomy.

Immortality is a common characteristic of cancers, but its origin and purpose are still unclear. Here we advance a karyotypic theory of immortality based on the theory that carcinogenesis is a form of speciation. Accordingly, cancers are generated from normal cells by random karyotypic rearrangements and selection for cancer-specific reproductive autonomy. Since such rearrangements unbalance long-established mitosis genes, cancer karyotypes vary spontaneously but are stabilized perpetually by clonal selections for autonomy. To test this theory we have analyzed neoplastic clones, presumably immortalized by transfection with overexpressed telomerase or with SV40 tumor virus, for the predicted clonal yet flexible karyotypes. The following results were obtained: (1) All immortal tumorigenic lines from cells transfected with overexpressed telomerase had clonal and flexible karyotypes; (2) Searching for the origin of such karyotypes, we found spontaneously increasing, random aneuploidy in human fibroblasts early after transfection with overexpressed telomerase; (3) Late after transfection, new immortal tumorigenic clones with new clonal and flexible karyotypes were found; (4) Testing immortality of one clone during 848 unselected generations showed the chromosome number was stable, but the copy numbers of 36% of chromosomes drifted ± 1; (5) Independent immortal tumorigenic clones with individual, flexible karyotypes arose after individual latencies; (6) Immortal tumorigenic clones with new flexible karyotypes also arose late from cells of a telomerase-deficient mouse rendered aneuploid by SV40 virus. Because immortality and tumorigenicity: (1) correlated exactly with individual clonal but flexible karyotypes; (2) originated simultaneously with such karyotypes; and (3) arose in the absence of telomerase, we conclude that clonal and flexible karyotypes generate the immortality of cancers.

Individual karyotypes at the origins of cervical carcinomas.

BACKGROUND: In 1952 Papanicolaou et al. first diagnosed and graded cervical carcinomas based on individual "abnormal DNA contents" and cellular phenotypes. Surprisingly current papilloma virus and mutation theories of carcinomas do not mention these individualities. The viral theory holds that randomly integrated, defective genomes of papilloma viruses, which are often untranscribed, cause cervical carcinomas with unknown cofactors 20-50 years after infection. Virus-free carcinomas are attributed to mutations of a few tumor-suppressor genes, especially the p53 gene. But the paradox of how a few mutations or latent defective viral DNAs would generate carcinomas with endless individual DNA contents, degrees of malignancies and cellular phenotypes is unsolved. Since speciation predicts individuality, we test here the theory that cancers are autonomous species with individual clonal karyotypes and phenotypes. This theory postulates that carcinogens induce aneuploidy. By unbalancing mitosis genes aneuploidy catalyzes chain reactions of karyotypic evolutions. Most such evolutions end with non-viable karyotypes but a few become new cancer karyotypes. Despite congenitally unbalanced mitosis genes cancer karyotypes are stabilized by clonal selections for cancer-specific autonomy. RESULTS: To test the prediction of the speciation theory that individual carcinomas have individual clonal karyotypes and phenotypes, we have analyzed here the phenotypes and karyotypes of nine cervical carcinomas. Seven of these contained papilloma virus sequences and two did not. We determined phenotypic individuality and clonality based on the morphology and sociology of carcinoma cells in vitro. Karyotypic individuality and clonality were determined by comparing all chromosomes of 20 karyotypes of carcinomas in three-dimensional arrays. Such arrays list chromosome numbers on the x-axis, chromosome copy numbers on the y-axis and the number of karyotypes arrayed on the z-axis. We found (1) individual clonal karyotypes and phenotypes in all nine carcinomas, but no virus-specific markers, (2) 1-to-1 variations between carcinoma-specific karyotypes and phenotypes, e.g. drug-resistance and cell morphology, (3) proportionality between the copy numbers of chromosomes and the copy numbers of hundreds of over- and under-expressed mRNAs, (4) evidence that tobacco-carcinogens induce cervical carcinomas via aneuploidy, consistent with the speciation theory. CONCLUSIONS: Since the individual clonal karyotypes of nine carcinomas correlated and co-varied 1-to-1 with complex individual transcriptomes and phenotypes, we have classical genetic and functional transcriptomic evidence to conclude that these karyotypes encode carcinomas - much like the clonal karyotypes that encode conventional species. These individual karyotypes explain the individual "DNA contents", the endless grades of malignancies and the complex individual transcriptomes and phenotypes of carcinomas.

Origin of metastases: subspecies of cancers generated by intrinsic karyotypic variations.

Conventional mutation theories do not explain (1) why the karyotypes of metastases are related to those of parental cancers but not to those of metastases of other cancers and (2) why cancers metastasize at rates that often far exceed those of conventional mutations. To answer these questions, we advance here the theory that metastases are autonomous subspecies of cancers, rather than mutations. Since cancers are species with intrinsically flexible karyotypes, they can generate new subspecies by spontaneous karyotypic rearrangements. This phylogenetic theory predicts that metastases are karyotypically related to parental cancers but not to others. Testing these predictions on metastases from two pancreatic cancers, we found: (1) Metastases had individual karyotypes and phenotypes. The karyotypes of metastases were related to, but different from, those of parental cancers in 11 out of 37 and 26 out of 49 parental chromosomal units. Chromosomal units are defined as intact chromosomes with cancer-specific copy numbers and marker chromosomes that are > 50% clonal. (2) Metastases from the two different cancers did not share chromosomal units. Testing the view that multi-chromosomal rearrangements occur simultaneously in cancers, as opposed to sequentially, we found spontaneous non-clonal rearrangements with as many new chromosomal units as in authentic metastases. We conclude that metastases are individual autonomous species differing from each other and parental cancers in species-specific karyotypes and phenotypes. They are generated from parental cancers by multiple simultaneous karyotypic rearrangements, much like new species. The species-specific individualities of metastases explain why so many searches for commonalities have been unsuccessful.