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Karyotype Alteration Generates the Neoplastic Phenotypes of SV40-infected Human and Rodent Cells

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Karyotype Alteration Generates the Neoplastic Phenotypes of SV40-infected Human and Rodent Cells

Mathew Bloomfield et al. Mol Cytogenet.

Abstract

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.

Keywords: Cancer-specific reproductive autonomy; Clonal karyotypes of cancers; Immortality; Individuality of cancer phenotypes and transcriptomes; Preneoplastic aneuploidy; Speciation theory of carcinogenesis.

Figures

Fig. 1
Fig. 1
Karyotypic theory of SV40 virus-induced neoplastic transformation. The karyotypic theory proposes that SV40 initiates carcinogenesis indirectly by inducing in infected cells preneoplastic aneuploidies at high rates (m1, in Fig. 1). Since aneuploidy destabilizes the karyotype by unbalancing thousands of genes, it catalyzes chain reactions of karyotypic and transcriptomic evolutions, also at high rates (m2, in Fig. 1). Eventually rare karyotypes evolve that encode cancer-specific autonomy of growth, at very low rates (m3, Fig. 1). The low probability of forming new autonomous cancer-species by random karyotypic and transcriptomic variations predicts the individuality of cancers. Although cancer karyotypes are congenitally aneuploid and thus unstable, they are stabilized or immortalized by selection for variants with cancer-specific autonomy. Owing to these inherent variations cancer karyotypes are heterogeneous within clonal margins (shaded in Fig. 1) [–102]. The resulting spreads of quasi-clonal karyotypes are defined by ‘karyotype arrays’ in which multiple individual karyotypes of the same cancers are compared (shown below in Figs. 5, 6, 7, 11, 12, 13, 16 and 17)
Fig. 2
Fig. 2
Phenotypes of human mesothelial cells three weeks after infection by SV40 compared to an uninfected control. A subconfluent culture of human mesothelial cells was infected with SV40 at a multiplicity of 10. In parallel an uninfected culture was maintained under the same conditions (see text). a A 120X magnification of the infected culture three weeks after infection shows highly increased cell density and “pre-transformed” cell morphologies [13], compared to the uninfected control shown in (b). It follows that SV40 transforms the morphology and raises the density of cultures of mesothelial cells shortly after infection
Fig. 3
Fig. 3
Cell morphologies of the immortal cell lines F1 and F4 derived from SV40-infected human mesothelial cells. The karyotypic cancer theory predicts that neoplastic karyotypes encode the individual phenotypes of SV40-transformed neoplastic clones. To test this prediction we have compared at 120X magnifications cultures of the immortal neoplastic cell lines F1 and F4, which arose from SV40-infected human mesothelial cells. It can be seen in the micrographs shown in (a) that the F1 line formed a dense monolayer of cells and in (b) that the F4 line formed a multilayer of cells. The micrographs also show that both lines consisted of round to oval polymorphic cells, and that the F4-cells were on average larger than the F1 cells. This result revealed phenotypic cellular similarity but sociological dissimilarity of the two cell lines
Fig. 4
Fig. 4
Karyotypes of the immortal cell lines F1 and F4 derived from a common culture of SV40-infected human mesothelial cells. To test, whether the karyotypes of F1and F4 would explain the individual but related phenotypes of F1 and F4, we compared their karyotypes. The karyotypes were prepared from metaphase chromosomes stained with chromosome-specific fluorescent colors following published procedures (Methods). As shown in (a), F1 has a hyper-diploid, aneuploid karyotype with 56 including 36 normal and 20 marker chromosomes. The karyotype shown in (b) indicates that F4 has a hypo-tetraploid karyotype with 81 including 56 normal and 25 marker chromosomes. However, a close comparison of the copy numbers of the ten F1-chromosomes, 2, 5, 10, 11, 12, 15, 18, 19, 20 and of one shared marker chromosome (the first on the list of marker chromosomes), with their F4-counterparts reveals that the F1-copy numbers are exactly duplicated in F4. This suggests that F4 probably originated from F1 by some form of karyotype duplication (see text)
Fig. 5
Fig. 5
Comparison of the karyotype arrays of the immortal F1 and F4 lines from SV40-infected human mesothelial cells. Karyotype arrays are three-dimensional tables of 20 karyotypes, which list the chromosome numbers of each karyotype on the x-axis, the copy numbers of each chromosome on the y-axis, and the number of karyotypes arrayed on the z-axis, as detailed in the text (Section I, Phenotypes and karyotypes of neoplastic clones from SV40-infected mesothelial cells). a and the attached table shows that the F1 line is hyper-diploid, consisting of 56 chromosomes that are 70 % to 100 % clonal. Accordingly the F1-chromosomes formed a quasi-clonal F1-array, which defines the F1 line. The non-clonal fraction of chromosomes included several partially clonal and several non-clonal marker chromosomes, indicative of ongoing karyotypic variation (see Fig. 1). b shows the F4 line is hypo-tetraploid consisting of 83 chromosomes which are 60–100 % clonal. Accordingly the F4-chromosomes formed a quasi-clonal F4-array, which is distinct from, but visibly related to that of the F1 line. The attached tables indicate that the differences between F1 and F4 include 19 F1-specific and 13 F4-specific clonal marker chromosomes. The tables also indicate that the copy numbers of 10 intact and one F4 marker chromosomes were exact duplications of the copy numbers of the corresponding F1-chromosomes (marked yellow in Fig. 5). In addition F1 and F4 shared two nullisomies of chromosomes 7 and 13. Moreover, several F1-chromosomes with non-duplicated copy numbers in F4 were increased in F4, but not exactly two-fold. This result thus indicates that the F4 line is a descendant of the F1 line generated by some form of tetraploidization (see text), rather than an independent clone
Fig. 6
Fig. 6
Comparison of the karyotype array of the F1 line with that of a puromycin-resistant derivative. To determine whether acquisition of resistance to puromycin was based on karyotypic variation, the karyotype array of a puromycin-resistant variant of F1was compared to that of the parental F1 clone. The array of the drug-resistant F1 shows a hyper-tetraploid karyotype consisting of 105 chromosomes that were 45–100 % clonal. Accordingly these chromosomes formed a quasi-clonal, resistant F1-specific array, which is distinct from, but visibly related to that of the parental F1 line, shown in Fig. 5a. Quantitative comparison of the chromosome copy numbers of the two F1 variants shown in the table of Fig. 6 revealed obvious similarities: 14 F1-chromsomes were exactly duplicated in the resistant variant and six others were increased approximately two-fold. It follows that the puromycin-resistant variant of F1 arose from the parental F1 line by an approximate tetraploidization; similar to how the above described near tetraploid F4 arose from the near diploid F1. This event was also associated with the acquisition of 29 new resistance-specific clonal marker chromosomes, and with the loss of three parental clonal marker chromosomes
Fig. 7
Fig. 7
Comparison of the karyotype array of the F4 line with that of a puromycin-resistant derivative. To determine whether resistance to puromycin of the F4 line was acquired by karyotypic variation, as was found in Fig. 6, the karyotype array of a puromycin-resistant F4 variant was compared to that of the parental F4 line. The array of the drug-resistant F4 shows a hypo-tetraploid karyotype consisting of 80 chromosomes that were 55–100 % clonal. Accordingly, these chromosomes formed a quasi-clonal array, which is distinct from, but visibly related to from the parental F4 line, shown in Fig. 5b. Specifically, the puromycin-resistant F4 variant differs from the parental line in the copy numbers of 15 of their 34-shared chromosomes (marked yellow in Fig. 7), the gain of two resistance-specific marker chromosomes and the loss of four parental marker chromosomes. We conclude that the F4 line acquired resistance to puromycin by karyotypic variation, as was the case with the puromycin-resistant variant of the F1 clone described in Fig. 6
Fig. 8
Fig. 8
Non-correlations between SV40 T-antigen and the cells of the immortal clones F1 and F4. To answer the question whether clonal cancer karyotypes are sufficient to generate and maintain neoplastic clones or are also dependent on T-antigen, we analyzed the immortal neoplastic clones F1 and F4 for the presence of viral T- antigen. For this purpose F1 and F4 cell cultures were reacted with mouse anti-T-antigen antibodies-linked to a green fluorescent dye and counter-stained with the blue fluorescent DNA dye, ‘DAPI,’ to detect nuclear DNA irrespective of T-antigen (Methods). a shows that about 30 % of the cells of the F1 line were T-antigen negative, while the remaining 70 % were heterogeneous for T-antigen expression ranging from very low to relatively high levels. b shows that about 50 % of the cells of F4 were T-antigen negative, whereas the rest of the cells were heterogeneous ranging from very low to relatively high T-antigen levels, similar to the F1 culture. We conclude that T-antigen is not necessary to maintain neoplastic transformation of F1 and F4 lines
Fig. 9
Fig. 9
Focus of transformed cells from a culture of rat lung cells three weeks after infection with SV40. This focus was one of 108 that arose in confluent secondary cultures of rat lung cells three weeks after infection of a primary culture with SV40 virus. The micrograph was taken at 120X magnification. Details of preparing the culture are described in the text
Fig. 10
Fig. 10
Individual cell morphologies of six focal colonies from cultured primary rat lung cells, three weeks after infection with SV40. To test the theory that the karyotypes of individual neoplastic clones encode individual phenotypes, we analyzed the cellular morphologies of six focal colonies from SV40-infected rat lung cells (see example in Fig. 9). As can be seen by the 120X magnification of cultures of the six focal colonies, F8 (a), F33 (b), F3 (c), F100 (d), F10 (e) and FC1 (f), all clones had individual cell morphologies. This result indicates that individual karyotypes, rather than common viral genes, encode the individual phenotypes of the distinct neoplastic clones
Fig. 11
Fig. 11
Karyotype arrays of six morphologically distinct focal colonies derived from SV40-infected rat lung cells: first pair of three. To test the theory that individual clonal karyotypes encode the individual phenotypes of neoplastic clones, the karyotype arrays the six morphologically distinct focal rat colonies F8, F33, F3, F100 and FC1, shown above in Fig. 10, were compared to each other in three separate Figures, namely 11, 12 and 13. a shows that F8 has a near-diploid karyotype with 41 chromosomes that were 85–100 % clonal. Accordingly, the F8-chromosomes formed a quasi-clonal F8-specific karyotype array. The non-clonal fraction of chromosomes included several partially clonal and several non-clonal marker chromosomes, indicative of ongoing karyotypic variation (see Fig. 1). b shows that F33 contained a pseudo-diploid karyotype with 42 chromosomes that were 70–100 % clonal. Accordingly, the F33 chromosomes also formed an individual quasi-clonal array that was different from that of F8. The individualities of these two karyotype arrays thus support the theory that individual karyotypes encode the individual phenotypes of neoplastic clones. (The karyotype arrays of the remaining four rat colonies are shown in Figs. 12 and 13)
Fig. 12
Fig. 12
Karyotype arrays of six morphologically distinct focal colonies from SV40-infected rat lung cells: second pair of three. To test the theory that individual clonal karyotypes encode the individual phenotypes, two more of the six morphologically distinct rat clones described in Fig. 10 were compared. a shows that F3 has a near-diploid karyotype of 41 chromosomes that were 70–100 % clonal. Accordingly, the F3 chromosomes formed an individual, quasi-clonal karyotype array, which was different from those of F8 and F33 shown in Fig. 11. A third of the F3 cells had near-tetraploid karyotypes that appeared to be duplications of the predominant near-diploid F3 karyotypes. b shows that F100 has a near-diploid karyotype with 43 chromosomes that were 95–100 % clonal. Accordingly the F100 chromosomes also formed a quasi-clonal F100 karyotype array, which was different from those of F3, F8 and F33. Thus the karyotypes of four distinct neoplastic rat clones support the theory that individual karyotypes encode the individual phenotypes of neoplastic clones
Fig. 13
Fig. 13
Karyotype arrays of six morphologically distinct focal colonies derived from SV40-infected rat lung cells: third pair of three. To test the theory that individual clonal karyotypes encode individual phenotypes, we analyzed and compared the arrays of a third pair of six individually distinct rat focal colonies. a shows that F10 has a near-diploid karyotype with an average number of 41 chromosomes that were between 70 and 100 % clonal. Accordingly they formed a quasi-clonal F10-specific array, which is different from those of the F8, F33, F3 and F100 colonies. The F10 clone also includes a minor (15 %) tetraploid variant, similar to that found in clone F3 described above (Fig. 12a). b shows that clone FC1 has a near triploid karyotype with 64 chromosomes that were between 55 and 100 % clonal. Accordingly they formed a quasi-clonal FC1-specific array, which differs from those of all five sister colonies described above and in Figs. 11 and 12. Thus the individual karyotypes of the six phenotypically distinct rat clones support the theory that individual karyotypes encode the individual phenotypes of neoplastic clones
Fig. 14
Fig. 14
Non-correlations between the cells of two SV40-induced neoplastic clones of rat lung cells and SV40 T-antigen. To test the prediction of the karyotypic theory that the clonal karyotypes of SV40-induced neoplastic clones are sufficient to generate and maintain neoplastic clones, independent of the viral T-antigen, we analyzed the cells of the SV40-induced neoplastic rat clones F3 and F100 (described in Figs. 10 and 12) for the presence of viral T-antigen with green-labeled antibodies, as described above for Fig. 8. a shows that about 30 % of the F3 cells were T-antigen negative, while the remaining 70 % were heterogeneous, expressing T-antigen between very low to relatively high levels. b shows that all cells of the neoplastic rat clone F100 were T-antigen negative under the conditions of our test (Methods). We concluded from the absence of detectable T-antigen in F100 and the absence or heterogeneous presence of T-antigen in F3 cells that T-antigen is not necessary to maintain neoplastic transformation of SV40-induced neoplastic rat clones, as predicted by the karyotypic theory
Fig. 15
Fig. 15
Distinct cell morphologies of four Individual focal colonies from SV40-infected mouse embryo cells. To test the theory that individual karyotypes encode neoplastic clones with individual phenotypes, we analyzed the cellular morphologies of four focal colonies that arose from SV40-infected mouse embryo cells. As seen by 125-fold magnification of cultures of four such colonies, F1 (a), F9 (b), F10 (c) and F11 (d), each clone had an individual and apparently clonal cell morphology. This result indicates that individual genotypes, rather than common SV40 genes encode the morphologies of these virus-induced colonies
Fig. 16
Fig. 16
Karyotype arrays of four morphologically distinct neoplastic colonies derived from SV40-infected mouse embryo cells: first two of four. To test the theory that individual clonal karyotypes encode the phenotypes of individual neoplastic clones from SV40-infected mouse cells, the karyotype arrays of the four focal mouse colonies F1, F9, F10 and F11, shown in Fig. 15 were compared to each other in two separate figures, namely 16 and 17. a shows that F1 has a near diploid karyotype with 39 chromosomes including three marker chromosomes, which were 88–100 % clonal. Accordingly the F1 chromosomes formed a quasi-clonal F1-specific karyotype array. b shows that F9 has a hypo-tetraploid karyotype with 76 chromosomes, which 60–95 % clonal. Accordingly the F9 chromosomes formed a quasi-clonal F9-specific karyotype array, which is different from that of F1. The 1-to-1 karyotype-phenotype correlation of F1 and F9 thus supports the theory that individual karyotypes, rather than common viral genes, encode the individual phenotypes of the SV40-induced neoplastic mouse clones
Fig. 17
Fig. 17
Karyotype arrays of four morphologically distinct neoplastic colonies derived from SV40-infected mouse embryo cells: second pair of two. To test the theory that individual clonal karyotypes encode individual phenotypes, the karyotypes of two of four mouse colonies, namely F10 and F11 were compared here. a shows that F10 has a hyper-tetraploid karyotype with 96 chromosomes that were 70–100 % clonal. Accordingly, the F10 chromosomes formed an F10-specific quasi-clonal array, which is different from those of F1 and F9. b shows that F11 has a near tetraploid karyotype with an average number of 78 chromosomes, which were 55–100 % clonal. Accordingly the F11 chromosomes also formed an individual quasi-clonal array, which is different from those of all three sister colonies, F1, F9, and F10 tested above. The individual karyotypes of the four phenotypically distinct mouse clones thus support the theory that individual karyotypes encode the individual phenotypes of neoplastic clones

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