Genetic and phenotypic instability are hallmarks of cancer cells, but their cause is not clear. The leading hypothesis suggests that a poorly defined gene mutation generates genetic instability and that some of many subsequent mutations then cause cancer. Here we investigate the hypothesis that genetic instability of cancer cells is caused by aneuploidy, an abnormal balance of chromosomes. Because symmetrical segregation of chromosomes depends on exactly two copies of mitosis genes, aneuploidy involving chromosomes with mitosis genes will destabilize the karyotype. The hypothesis predicts that the degree of genetic instability should be proportional to the degree of aneuploidy. Thus it should be difficult, if not impossible, to maintain the particular karyotype of a highly aneuploid cancer cell on clonal propagation. This prediction was confirmed with clonal cultures of chemically transformed, aneuploid Chinese hamster embryo cells. It was found that the higher the ploidy factor of a clone, the more unstable was its karyotype. The ploidy factor is the quotient of the modal chromosome number divided by the normal number of the species. Transformed Chinese hamster embryo cells with a ploidy factor of 1.7 were estimated to change their karyotype at a rate of about 3% per generation, compared with 1.8% for cells with a ploidy factor of 0.95. Because the background noise of karyotyping is relatively high, the cells with low ploidy factor may be more stable than our method suggests. The karyotype instability of human colon cancer cell We conclude that aneuploidy is sufficient to explain genetic instability and the resulting karyotypic and phenotypic heterogeneity of cancer cells, independent of gene mutation. Because aneuploidy has also been proposed to cause cancer, our hypothesis offers a common, unique mechanism of altering and simultaneously destabilizing normal cellular phenotypes.

Only two avian oncogenic viruses specifically cause acute leukaemias yet do not transform chicken fibroblasts in culture: E26, which causes erythroblastosis and a low level of concomitant myeloblastosis in chickens, and avian myeloblastosis virus (AMV), which causes myeloblastosis exclusively. Both viruses are replication-defective and share a sequence termed myb (also known as amv) which is unrelated to essential virion genes and is therefore thought to be part of the transforming onc genes of these viruses. However, the genetic structure of the two viruses differs. E26 has a genomic RNA of 5.7 kilobases (kb) and encodes a 135,000 molecular weight gag-related protein (p135) with probable transforming function. We show here by in vitro translation that the 5.7-kb E26 RNA directs the synthesis of p135. Oligonucleotide analysis indicates that E26 RNA contains an internal 0.8-kb subset of the 1.2-kb AMV-related sequence (mybA), termed mybE. A 2.46-kb molecular clone prepared from cDNA transcribed in vitro from E26 RNA contained an E26 transformation-specific (ets) sequence flanked by mybE and an env-related sequence. A complete DNA sequence of this clone indicates that the 1.5-kb ets sequence extends the open reading frame of mybE for 491 amino acids. Thus, the p135 gene of E26 is a genetic hybrid of three distinct elements, approximately 1.2 kb derived from the 5' region of the retroviral gag gene, mybE and the ets sequence, linked in the order 5'-delta gag-mybE-ets-3'. The myeloid leukaemogenicity shared by E26 and AMV correlates with the common myb sequence, while the distinct erythroid leukaemogenicity of E26 correlates with ets and the E26-specific linkage of myb to delta gag.

Abstract. The 60-70S RNAs of several transforming and nontransforming avian tumor viruses have different electrophoretic mobilities. The RNA of transforming viruses contains two electrophoretically separable subunit classes: a and b. The relative concentrations of these subunits vary with the virus strain. Avian leukosis viruses and nontransforming derivatives of a sarcoma virus lack subunits of class a. It is suggested that the presence of the class a subunit is related to the transforming ability for fibroblasts of the virus.

No abstract

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 errorprone, 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. Carcinogenesis is a very rare process in which a normal cell is converted to a cancer cell via multiple "steps" or "stages" of cellular evolution, which correspond to various pre-neoplastic and neoplastic phenotypes and genotypes. 1-4 The clinical and biological phenotypes of the multiple steps of carcinogenesis include hyperplasia, dysplasia, abnormal morphology, anaplasia or dedifferentiation, drug-and multi-drug resistance, immortality, altered histocompatibility including even transplantability to some heterologous species and susceptibility to some heterologous viruses, abnormal metabolism, autonomous growth, invasiveness, and metastasis. 1,5-12 Based on various genetic and cytogenetic markers, including structurally altered or marker chromosomes, most cancers are clonal, i.e., derived from a single cell. 3,13-15 However, a minority is polyclonal. 1,16,17 Despite their clonal origin, "the structure and behavior of tumors are determined by numerous, abnormal characters that, within wide limits, are independently variable, capable of highly varied combinations and assortments and liable to independent progression." 18,19 As a result of this "genetic instability" 3 hardly any two cells from a given cancer are ever the same. 1,20-26,62,137 Indeed, the specific karyotypes and phenotypes of individual cancer cells can v...

The 28S RNA of the defective avian acute leukemia virus MC29 contains two sets of sequences: 60% are hybridized by DNA complementary to other avian tumor virus RNAs (group-specific cDNA) and 40% are hybridized only by MC29-specific cDNA. Specific and group-specific sequences of viral RNA, defined in terms of their large RNase TI-resistant oligonucleotides, were located on a map of all large T, oligonucleotides of viral RNA. Oligonucleotides representing MC2-specific sequences of viral RNAmnapped between 0.4 and 0.7 unit from the 3'-poly(A) end. Oligonucleotides of groupspecific sequences mapped between 0 and 0.4 and between 0.7 add I map unit. Cell-free translation of viral RNA yielded three proteins with approximate molecular weights of 120,000,356,000, and 37,000, termed P120me P5i, and P37mc. P120me contained both MC29specific peptiies and serological determinants and peptides of the conserved, internal group-specific antigens of avian tumor viruses. P120me is translated only from ful~length 28S RNA. Furthermore, MC29 RNA contains sequences related to the groupspecific antigen gene (gag), near the 5' end, which are folowed by MC29specific sequences. We conclude that this protein is translated from the 5' 60% of the RNA, and that it includes a segment translated from the specific sequences. It is suggested that the transforming (onc) gene of MC29 may consists of the specific and some group-specific RNA sequences and that Pl2rnc, which is also found in transformed cells, may be the onc gene product. MC29 is an avian RNA tumor virus that causes acute leukemia and carcinoma and also transforms fibroblasts in culture (1-4). The transforming or onc (5) gene of MC29 has not been defined genetically or biochemically. The viral genome is a 28S RNA (5700 nucleotides) that is identified as being MC29-specific by its absence from pure helper virus and because the sequence of 28S RNA is conserved when propagated with different helper viruses (6)(7)(8) However, if the specific sequences of MC29 are contiguous and if they code for a specific protein, they are candidates for a MC29 onc gene analogous to the src gene of RSV. Therefore we identified and located the MC29-specific sequences on the viral 28S RNA and then investigated the proteins encoded by these sequences. Preliminary work has been described recently (8). RESULTSMapping MC29-Specific and Group-Specific Sequences of MC29 RNA. Due to its defectiveness, MC29 virus can only be propagated in the presence of a helper virus. The helper virus used here was ring-neck pheasant virus (RPV) (4). The 50-70S RNA extracted from a mixture of MC29 and its helper contains two distinct monomer RNA species, a 34S helper virus RNA and a 28S MC29 RNA (6). The 28S MC29 RNA has been electrophoretically isolated and its RNase T1-resistant oligonucleotides have been analyzed by fingerprinting (Fig. 1A). The compositions, of the RNase A-resistant fragments of each oligonucleotide have been reported (6-8) and were extended and revised here as described in the legend of Fig. 1. To ...

The hypothesis that human immunodeficiency virus (HIV) is a new, sexually transmitted virus that causes AIDS has been entirely unproductive in terms of public health benefits. Moreover, it fails to predict the epidemiology of AIDS, the annual AIDS risk and the very heterogeneous AIDS diseases of infected persons. The correct hypothesis must explain why: (1) AIDS includes 25 previously known diseases and two clinically and epidemiologically very different epidemics, one in America and Europe, the other in Africa; (2) almost all American (90%) and European (86%) AIDS patients are males over the age of 20, while African AIDS affects both sexes equally; (3) the annual AIDS risks of infected babies, intravenous drug users, homosexuals who use aphrodisiacs, hemophiliacs and Africans vary over 100-fold; (4) many AIDS patients have diseases that do not depend on immunodeficiency, such as Kaposi's sarcoma, lymphoma, dementia and wasting; (5) the AIDS diseases of Americans (97%) and Europeans (87%) are predetermined by prior health risks, including long-term consumption of illicit recreational drugs, the antiviral drug AZT and congenital deficiencies like hemophilia, and those of Africans are Africa-specific. Both negative and positive evidence shows that AIDS is not infectious: (1) the virus hypothesis fails all conventional criteria of causation; (2) over 100-fold different AIDS risks in different risk groups show that HIV is not sufficient for AIDS; (3) AIDS is only 'acquired,' if at all, years after HIV is neutralized by antibodies; (4) AIDS is new but HIV is a long-established, perinatally transmitted retrovirus; (5) alternative explanations disprove all assumptions and anecdotal cases cited in support of the virus hypothesis; (6) all AIDS-defining diseases occur in matched risk groups, at the same rate, in the absence of HIV; (7) there is no common, active microbe in all AIDS patients; (8) AIDS manifests in unpredictable and unrelated diseases; and (9) it does not spread randomly between the sexes in America and Europe. Based on numerous data documenting that drugs are necessary for HIV-positives and sufficient for HIV-negatives to develop AIDS diseases, it is proposed that all American/European AIDS diseases, that exceed their normal background, result from recreational and anti-HIV drugs. African AIDS is proposed to result from protein malnutrition, poor sanitation and subsequent parasitic infections. This hypothesis resolves all paradoxes of the virus-AIDS hypothesis. It is epidemiologically and experimentally testable and provides a rational basis for AIDS control.

The mutation rates of cancer cells to drug and multidrug resistance are paradoxically high, i.e., 10 ؊3 to 10 ؊6 , compared with those altering phenotypes of recessive genes in normal diploid cells of about 10 ؊12 . Here the hypothesis was investigated that these mutations are due to chromosome reassortments that are catalyzed by aneuploidy. Aneuploidy, an abnormal number of chromosomes, is the most common genetic abnormality of cancer cells and is known to change phenotypes (e.g., Down's syndrome). Moreover, we have shown recently that aneuploidy autocatalyzes reassortments of up to 2% per chromosome per mitosis because it unbalances spindle proteins, even centrosome numbers, via gene dosage. The hypothesis predicts that a selected phenotype is associated with multiple unselected ones, because chromosome reassortments unbalance simultaneously thousands of regulatory and structural genes. It also predicts variants of a selected phenotype based on variant reassortments. To test our hypothesis we have investigated in parallel the mutation rates of highly aneuploid and of normal diploid Chinese hamster cells to resistance against puromycin, cytosine arabinoside, colcemid, and methotrexate. The mutation rates of aneuploid cells ranged from 10 ؊4 to 10 ؊6 , but no drug-resistant mutants were obtained from diploid cells in our conditions. Further selection increased drug resistance at similar mutation rates. Mutants selected from cloned cells for resistance against one drug displayed different unselected phenotypes, e.g., polygonal or fusiform cellular morphology, flat or threedimensional colonies, and resistances against other unrelated drugs. Thus our hypothesis offers a unifying explanation for the high mutation rates of aneuploid cancer cells and for the association of selected with unselected phenotypes, e.g., multidrug resistance. It also predicts drug-specific chromosome combinations that could become a basis for selecting alternative chemotherapy against drugresistant cancer.