Maintenance of Normal in situ Chromosomal Features in Long-Term Tissue Cultures

Yerganian, George; Leonard, Mary J.

doi:10.1126/science.133.3464.1600

Cited by 62 publications

(11 citation statements)

References 7 publications

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“…Between 4 and 6 million CH cells, the equivalent of 0.5-1 confluent 10-cm Petri dish, were dissociated with trypsin, resuspended in Ϸ0.3 ml of PBS, and injected into in the upper hind leg of 6-9-month-old syngeneic, male CHs (34). Tumors of 1-1.5 cm were explanted at the time of diagnosis, minced with scalpels, dissociated into single cells with trypsin, and then cultured until a confluent Petri dish was obtained for the analysis of metaphases as described (24).…”

Section: Methodsmentioning

confidence: 99%

Specific aneusomies in Chinese hamster cells at different stages of neoplastic transformation, initiated by nitrosomethylurea

Fabarius

Willer

Yerganian

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Aneuploidy is ubiquitous in cancer, and its phenotypes are inevitably dominant and abnormal. In view of these facts we recently proposed that aneuploidy is sufficient for carcinogenesis generating cancerspecific aneusomies via a chain reaction of autocatalytic aneuploidizations. According to this hypothesis a carcinogen initiates carcinogenesis via a random aneuploidy. Aneuploidy then generates transformation stage-specific aneusomies and further random aneusomies autocatalytically, because it renders chromosome segregation and repair mechanisms error-prone. The hypothesis predicts that several specific aneusomies can cause the same cancers, because several chromosomes also cooperate in normal differentiation. Here we describe experiments on the Chinese hamster (CH) that confirm this hypothesis. (i) Random aneuploidy was detected before transformation in up to 90% of CH embryo cells treated with the carcinogen nitrosomethylurea (NMU). (ii) Several specific aneusomies were found in 70 -100% of the aneuploid cells from colonies transformed with NMU in vitro and from tumors generated by NMUtransformed cells in syngeneic animals. Among the aneuploid in vitro transformed cells, 79% were trisomic for chromosome 3, and 59% were monosomic for chromosome 10, compared with 8% expected for random distribution of any aneusomy among the 12 CH chromosomes. Moreover, 52% shared both trisomy 3 and monosomy 10 compared with 0.6% expected for random distribution of any two aneusomies. Among the tumor cells, 65% were trisomic for chromosome 3, 51% were trisomic for chromosome 5, and 30% shared both trisomies. Aneuploid cells without these specific aneusomies may contain minor transformation-specific aneusomies or may be untransformed. (iii) Random aneusomies and structurally altered chromosomes increased with the generations of transformed cells to the point where their origins became unidentifiable in tumors. We conclude that specific aneusomies are necessary for carcinogenesis.

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Section: Methodsmentioning

confidence: 99%

Specific aneusomies in Chinese hamster cells at different stages of neoplastic transformation, initiated by nitrosomethylurea

Fabarius

Willer

Yerganian

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…Subconfluent cultures of CHE cells, derived from a single male, 18-day embryo (71), that had been kept growing for one or two consecutive passages in culture were incubated for 2-3 hr with 0.1 g͞ml of colcemid (GIBCO͞BRL). The cells were then rinsed with phosphate-buffered physiological saline, dissociated with trypsin at 37°C, mixed with 1 ml complete culture medium, and centrifuged for 6 min at 500 rpm at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…The CHE in vitro system also allows a distinction between chemical and spontaneous transformation, because spontaneous transformation occurs at a much lower frequency and, on average, only many more cell divisions later (19,(71)(72)(73) than chemical transformation (20,66,68). We have also studied the chromosomes of spontaneously transformed CHE cells.…”

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confidence: 99%

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Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Yerganian

Duesberg

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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162

111

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Aneuploidy or chromosome imbalance is the most massive genetic abnormality of cancer cells. It used to be considered the cause of cancer when it was discovered more than 100 years ago. Since the discovery of the gene, the aneuploidy hypothesis has lost ground to the hypothesis that mutation of cellular genes causes cancer. According to this hypothesis, cancers are diploid and aneuploidy is secondary or nonessential. Here we reexamine the aneuploidy hypothesis in view of the fact that nearly all solid cancers are aneuploid, that many carcinogens are nongenotoxic, and that mutated genes from cancer cells do not transform diploid human or animal cells. By regrouping the gene pool-as in speciation-aneuploidy inevitably will alter many genetic programs. This genetic revolution can explain the numerous unique properties of cancer cells, such as invasiveness, dedifferentiation, distinct morphology, and specific surface antigens, much better than gene mutation, which is limited by the conservation of the existing chromosome structure. To determine whether aneuploidy is a cause or a consequence of transformation, we have analyzed the chromosomes of Chinese hamster embryo (CHE) cells transformed in vitro. This system allows (i) detection of transformation within 2 months and thus about 5 months sooner than carcinogenesis and (ii) the generation of many more transformants per cost than carcinogenesis. To minimize mutation of cellular genes, we have used nongenotoxic carcinogens. It was found that 44 out of 44 colonies of CHE cells transformed by benz[a]pyrene, methylcholanthrene, dimethylbenzanthracene, and colcemid, or spontaneously were between 50 and 100% aneuploid. Thus, aneuploidy originated with transformation. Two of two chemically transformed colonies tested were tumorigenic 2 months after inoculation into hamsters. The cells of transformed colonies were heterogeneous in chromosome number, consistent with the hypothesis that aneuploidy can perpetually destabilize the chromosome number because it unbalances the elements of the mitotic apparatus. Considering that all 44 transformed colonies analyzed were aneuploid, and the early association between aneuploidy, transformation, and tumorigenicity, we conclude that aneuploidy is the cause rather than a consequence of transformation.

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“…NHDF, (Promocell GmbH, Heidelberg Germany), CHE, derived from embryos of an inbred line of animals described previously, 21 and IMF, (American Type Culture Collection, Manassas, VA, USA) were grown in RPMI 1640 medium with 10% fetal calf serum, 1% calf serum, antibiotics, and antimycotics (Invitrogen Inc., Groningen, The Netherlands) and treated with 5, 10, or 20 mM of imatinib (provided by Novartis, Basel, Switzerland).…”

Section: Primary Cellsmentioning

confidence: 99%

Induction of centrosome and chromosome aberrations by imatinib in vitro

et al. 2005

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Imatinib (STI571, Gleevec/Glivec) is a potent selective tyrosine kinase inhibitor and is used successfully in the treatment of chronic myeloid leukemia (CML). While karyotype alterations, in addition to the Philadelphia chromosome, are a common phenomenon of progressing CML, the observation of BCR-ABL-negative leukemic clones with distinct aberrant karyotypes under an imatinib regimen is not yet understood. Here we test the hypothesis that such tumor clones may be induced de novo from normal cells by imatinib. In vitro experiments with varying drug concentrations (5-20 lM) were performed on normal human dermal fibroblasts (NHDF), Chinese hamster embryonal and Indian muntjak fibroblasts. After 3 weeks of treatment, analysis of cell cultures by centrosome immunostaining and conventional cytogenetics revealed that imatinib induced centrosome and chromosome aberrations in all cultures in a significant dose-dependent and species-independent manner. Moreover, the results of NHDF long-term culture experiments demonstrated that aberrant phenotypes, emerging under imatinib treatment for 12 weeks, were not reversible after prolonged propagation omitting the drug. These observations suggest a causative role of imatinib in the origin of centrosome and karyotype aberrations (genetic instability) and thus may explain the emergence of clonal chromosomal abnormalities in BCR-ABL-negative progenitor cells under imatinib therapy.

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Maintenance of Normal in situ Chromosomal Features in Long-Term Tissue Cultures

Cited by 62 publications

References 7 publications

Specific aneusomies in Chinese hamster cells at different stages of neoplastic transformation, initiated by nitrosomethylurea

Specific aneusomies in Chinese hamster cells at different stages of neoplastic transformation, initiated by nitrosomethylurea

Aneuploidy correlated 100% with chemical transformation of Chinese hamster cells

Induction of centrosome and chromosome aberrations by imatinib in vitro

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