Continuous growth of telomerase-immortalised fibroblasts: How long do cells remain normal?

Waarde-Verhagen, van Maria; Kampinga, Harm H.; Linskens, Maarten H.K.

doi:10.1016/j.mad.2005.08.008

Cited by 12 publications

(11 citation statements)

References 9 publications

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“…Such high mutation frequencies represent a caution in the use of immortalized cells for mutagenesis studies. Although some reports claim that hTERT-immortalization is nonmutagenic and maintains diploidy during extended culture (32,33), our observations, and those of others (34)(35)(36), suggest instead that continued in vitro proliferation under ambient oxygen can itself be mutagenic.…”

Section: Discussioncontrasting

confidence: 69%

Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

Reid-Bayliss

Arron

Loeb

et al. 2016

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

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Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.

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Section: Discussioncontrasting

confidence: 69%

Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

Reid-Bayliss

Arron

Loeb

et al. 2016

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

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“…However, it has also been reported that an hTERT transduced hNPC line gained an abnormal karyotype and formed neuroblastoma-like tumors after transplantation [46]. Furthermore, recent data indicate that other hTERT immortalized somatic cell lines show changes in cell cycle regulatory genes and karyotypes after prolonged culturing [47][48][49][50]. Using LIF treatment of hNPC to activate telomerase and thereby allow extended proliferative capacity may provide an alternative means for massive expansion without the risk associated with changing the genome by introducing the hTERT gene or other immortalizing agents.…”

Section: Discussionmentioning

confidence: 99%

Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro

Wright

Prowse

Wallace

et al. 2006

Experimental Cell Research

128

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Isolation of a true self-renewing stem cell from the human brain would be of great interest as a reliable source of neural tissue. Here, we report that human fetal cortical cells grown in epidermal growth factor expressed low levels of telomerase and telomeres in these cultures shortened over time leading to growth arrest after 30 weeks. Following leukemia inhibitory factor (LIF) supplementation, growth rates and telomerase expression increased. This was best demonstrated following cell cycle synchronization and staining for telomerase using immunocytochemistry. This increase in activity resulted in the maintenance of telomeres at approximately 7 kb for more than 60 weeks in vitro. However, all cultures displayed a lack of oligodendrotye production, decreases in neurogenesis over time and underwent replicative senescence associated with increased expression of p21 before 70 weeks in vitro. Thus, under our culture conditions, these cells are not stable, multipotent, telomerase expressing self-renewing stem cells. They may be more accurately described as human neural progenitor cells (hNPC) with limited lifespan and bi-potent potential (neurons/astrocytes). Interestingly, hNPC follow a course of proliferation, neuronal production and growth arrest similar to that seen during expansion and development of the human cortex, thus providing a possible model neural system. Furthermore, due to their high expansion potential and lack of tumorogenicity, these cells remain a unique and safe source of tissue for clinical transplantation.

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“…The Human fibroblast cell line K21 immortalized with hTERT (32) was a kind gift from Prof. M.H.K. Linskens, University of Groningen, The Netherlands.…”

Section: Methodsmentioning

confidence: 99%

Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

Biela

Coste

Culard

et al. 2014

Nucleic Acids Research

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DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity.

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Continuous growth of telomerase-immortalised fibroblasts: How long do cells remain normal?

Cited by 12 publications

References 9 publications

Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

Why Cockayne syndrome patients do not get cancer despite their DNA repair deficiency

Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro

Zinc finger oxidation of Fpg/Nei DNA glycosylases by 2-thioxanthine: biochemical and X-ray structural characterization

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