Culture conditions induce the appearance of immortalized C3H mouse cell lines

Curatolo, L.; Erba, Eugenio; Morasca, L.

doi:10.1007/bf02619607

Cited by 24 publications

(15 citation statements)

References 16 publications

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“…Rare cells grow past this barrier, termed crisis, to form p e r m a n e n t cell lines that are capable of growing in culture indefinitely. Rodent cells have proven to be the m o s t amenable to the study of this process because they immortalize in culture efficiently and spontaneously (Todaro and Green 1963;Meek et al 1977;Curatolo et al 1984). This is in contrast to normal h u m a n and avian primary fibroblasts, which have not been observed to i m m o r t a l i z e spontaneously in culture (Hayflick 1965;Smith and PereiraSmith 1990).…”

Section: Discussionmentioning

confidence: 99%

“…The frequency at which cells escape senescence appears to be species dependent. Whereas rodent cells have been observed to undergo spontaneous immortalization quite efficiently (Todaro and Green 1963;Meek et al 1977;Curatolo et al 1984), normal human and avian cells have rarely, if ever, been shown to be capable of spontaneous immortalization (Hayflick 1965;Smith and Pereira-Smith 1990).…”

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confidence: 99%

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p53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts.

Harvey¹,

Levine²

1991

Genes & Development

424

257

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It has been shown previously that mutant p53 can act as an immortalizing gene when cotransfected into primary rat embryo fibroblasts along with a selectable marker. To determine whether a mutation at the p53 locus is a common event in the pathways leading to spontaneous cellular immortalization, 11 clonally derived BALB/c murine embryo fibroblast lines were established by passage on a 3T3 schedule and examined for p53 alterations. By the following criteria, all 11 independently established lines contain at least one mutant allele of p53. Seven of these lines have a PAb240-reactive p53 species and exhibit an extended p53 half-life as determined by pulse-chase analysis. The p53 protein species in a subset of these lines is also capable of complex formation with the constitutive heat shock protein hsc70, p53 cytoplasmic DNAs (cDNAs) from several of these lines have been cloned by reverse transcription of cytoplasmic RNA followed by PCR amplification, and the mutations have been mapped by DNA sequence analysis. Point mutation in conserved domains of p53 appears to be a common alteration in these lines, although one established line carries a 24-bp in-frame deletion of p53. The remaining four cell lines do not express detectable p53 protein. For each line there is a different molecular event underlying the lack of p53 expression: (1) deletion of at least the first 6 exons of both p53 alleles; (2) expression of a single p53 mRNA encoding a stop codon at amino acid position 173; (3) no detectable p53 mRNA; and (4) greatly diminished expression of p53 mRNA. These findings indicate that p53 alteration commonly occurs in spontaneously immortalized BALB/c mouse embryo fibroblasts passaged on a 3T3 schedule and, therefore, may be an important event for the immortalization process.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

p53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts.

Harvey¹,

Levine²

1991

Genes & Development

424

257

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“…At present, the reports on malignant transformation of cultured human cells in vitro are rare (6-9,12,13), while malignant transformation in vitro could be observed in embryonic cells from mice (14)(15)(16). One hypothesis to explain this phenomenon is that the number of mutational events required to confer immortality on rodent cells is fewer than the number required for human and avian cells (or the rates of mutation are different) (17).…”

Section: Discussionmentioning

confidence: 99%

Tumor suppressor gene alterations of spontaneously malignant transformed cells from human embryonic muscle in vitro

Li²,

Zheng³

et al. 2010

Oncol Rep

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Abstract.Recent research has shown that mesenchymal stem cells (MSCs) which were cultured for long time could transform malignantly, the transformation mechanism is not clear yet, it might be associated with the activation of oncogenes and inactivation of tumor suppressor genes. In our initial investigation, we found that the cells arising from human embryonic muscle could spontaneously transform into malignancy in vitro and we obtained 6 immortalized cell lines. In this study, polymerase chain reaction (PCR) was used to assay several tumor suppressor genes of these cell lines, and homozygous deletions within chromosomal band 9p2l including MTAP (methylthioadenosine phosphorylase), p16 and p15 were detected. PCR products of p53 exons 7 and 8 of these novel tumor cell lines were assayed by sequencing, and the results showed high prevalence of mutations in these regions, the mutation rate reached as high as 8% in exon 7 and 14% in exon 8, and all of them were point mutations, the intron 7 changed more significantly, including piece deletion, insertion, frameshift and point mutation, it showed almost no similarity to that of the wt p53 sequence, that was totally different from other p53 mutation data published. All the mutation sequences were identical in 6 cell lines, this suggest that there may be a common mutation mechanism and strong selective advantage in these novel tumor cell lines over long-term culture. In conclusion, our research shows that the inactivation of tumor suppressor genes may play an important role in the process of malignant transformation of embryonic muscle cells in vitro. IntroductionThe development of tumors is generally accepted to be a multistep process in which alterations in oncogenes and tumor suppressor genes play an important role (1,2). The process of carcinogenesis involves the gain of oncogene activity and the loss of tumor suppressor gene function, such as Rb, p53 and cyclin-dependent kinase inhibitor (CDKI) family (3-5). The aberrant gene could interfere in many cell funtions, such as cell proliferation, differentiation and apoptosis. The accumulative mutations constitute the base of cell malignant transformation in gene level. The acquired capabilities of tumor cells include their ability to proliferate continuously ignoring apoptosis or growth-inhibitory signals, generating their own mitogenic signals.At present, the reports on the malignant transformation of human cell in vitro are rare. Recent research has found that mesenchymal stem cells (MSCs) cultured for long time in vitro could lead to malignant transformation, but the mechanism is not very clear. The activation of oncogenes and inactivation of tumor suppressor genes are considered to be one of the mechanisms (6-9). We have found that human embryonic muscle cells which were cultured in vitro longterm could become spontaneously immortalized, and we obtained 6 novel malignant cell lines, this new type of cell lines were named human embryonic muscle-derived malignant transformed cells (hEMTCs). These hEMTCs could be pass...

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“…At the first passage, cells were grown in 25-cm2 plastic flasks (Corning, France) as follows: the culture medium (0.4 ml/cm2) was renewed only at subculture, cells were split 1:2 at confluency, which is defined as a decrease in mitotic index [Curatolo et al, 1984], This culture procedure gave rise to three immortalized sister pop ulations from the parental line, namely A, B and C. The three immortalized lines appeared during the late 'phase three' (i.e. the last phase of a normal life span).…”

Section: Cells and Culture Methodsmentioning

confidence: 99%

“…In a previous study we found that slight differences in culture procedures produce different effects on normal C3H mouse em bryo cells, determining the success of 'spon taneous' immortalization and transforma tion [Curatolo et al, 1984], In view of the reproducibility of our culture method for in ducing immortalization (which was achieved in about 85% of the cell populations studied [Curatolo et al, 1984], this study was under taken to characterize three transformed lines derived from a C3H normal embryo fibro blastic primary culture [Curatolo et al, 1980].…”

Section: Introductionmentioning

confidence: 99%

Characterization of Transformed Cell Lines Evolving from a Normal C3H Line

Curatolo¹,

Frascotti²,

Ubezio³

et al. 1988

Pathobiology

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The development of transformed cell lines evolving from an embryo fibroblastic C3H primary culture was followed before and after the ageing crisis using different techniques. By flow cytometry, alteration of subpopulations having different DNA content and altered metabolic activity was observed after the crisis, with the trend to assume a near tetraploid DNA index at higher passages. The fibrin clot retractile activity was lost in all cases during the ageing crisis, but the outcome did not present uniform values of growth characteristics or chromosome number and tumorigenicity appeared to be a nonstable property of the transformed cell lines.

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Culture conditions induce the appearance of immortalized C3H mouse cell lines

Cited by 24 publications

References 16 publications

p53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts.

p53 alteration is a common event in the spontaneous immortalization of primary BALB/c murine embryo fibroblasts.

Tumor suppressor gene alterations of spontaneously malignant transformed cells from human embryonic muscle in vitro

Characterization of Transformed Cell Lines Evolving from a Normal C3H Line

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