Mutant frequencies and spectra depend on growth state and passage number in cells cultured from transgenic lacZ-plasmid reporter mice.

2.50
Hdl Handle:
http://hdl.handle.net/10029/6920
Title:
Mutant frequencies and spectra depend on growth state and passage number in cells cultured from transgenic lacZ-plasmid reporter mice.
Authors:
Busuttil, Rita A; Rubio, Miguel; Dollé, Martijn E T; Campisi, Judith; Vijg, Jan
Abstract:
Transgenic mice harboring the lacZ gene within a plasmid that can be recovered and amplified in Escherichia coli, to establish mutant frequencies and spectra, have provided crucial insights into the relationships between mutations, cancer and aging in vivo. Here, we use embryonic fibroblasts from transgenic lacZ-plasmid reporter mice to determine the relationship between cell proliferation in culture and mutations induced by ultraviolet (UV) light. A single dose of 2.5J/m2 of UVC to actively proliferating cells caused an approximately eight-fold increase in mutant frequency 24 h after irradiation. Identically treated quiescent cells showed a two-fold increase in mutant frequency. Thus, whereas proliferation facilitated the acquisition of mutations, it was not an absolute requirement. Characterization of the UV-induced mutations indicated that the lower mutant frequency in quiescent cells was due mainly to a reduction in point mutations; size-change mutations, indicative of translocations or deletions, were relatively unaffected by the growth state of the cells. To investigate long-term genomic stability after UVC-induced damage, we monitored the lacZ locus in irradiated cells passaged for many generations in culture. The results indicated the emergence of jackpot mutations of rapidly changing frequency, most likely reflecting the successive emergence and decline of dominant cell clones during long-term culture. These findings show that the lacZ-plasmid locus is a valid reporter for studying induced mutations in short-term cultures of both quiescent and proliferating fibroblasts. In long-term cultures, the locus is less suitable for studying induced mutations owing to the instability of the cell population.
Citation:
DNA Repair (Amst.) 2006, 5(1):52-60
Issue Date:
5-Jan-2006
URI:
http://hdl.handle.net/10029/6920
DOI:
10.1016/j.dnarep.2005.07.006
PubMed ID:
16126462
Type:
Article
Language:
en
ISSN:
1568-7864
Appears in Collections:
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Full metadata record

DC FieldValue Language
dc.contributor.authorBusuttil, Rita A-
dc.contributor.authorRubio, Miguel-
dc.contributor.authorDollé, Martijn E T-
dc.contributor.authorCampisi, Judith-
dc.contributor.authorVijg, Jan-
dc.date.accessioned2007-01-05T09:08:21Z-
dc.date.available2007-01-05T09:08:21Z-
dc.date.issued2006-01-05-
dc.identifier.citationDNA Repair (Amst.) 2006, 5(1):52-60en
dc.identifier.issn1568-7864-
dc.identifier.pmid16126462-
dc.identifier.doi10.1016/j.dnarep.2005.07.006-
dc.identifier.urihttp://hdl.handle.net/10029/6920-
dc.description.abstractTransgenic mice harboring the lacZ gene within a plasmid that can be recovered and amplified in Escherichia coli, to establish mutant frequencies and spectra, have provided crucial insights into the relationships between mutations, cancer and aging in vivo. Here, we use embryonic fibroblasts from transgenic lacZ-plasmid reporter mice to determine the relationship between cell proliferation in culture and mutations induced by ultraviolet (UV) light. A single dose of 2.5J/m2 of UVC to actively proliferating cells caused an approximately eight-fold increase in mutant frequency 24 h after irradiation. Identically treated quiescent cells showed a two-fold increase in mutant frequency. Thus, whereas proliferation facilitated the acquisition of mutations, it was not an absolute requirement. Characterization of the UV-induced mutations indicated that the lower mutant frequency in quiescent cells was due mainly to a reduction in point mutations; size-change mutations, indicative of translocations or deletions, were relatively unaffected by the growth state of the cells. To investigate long-term genomic stability after UVC-induced damage, we monitored the lacZ locus in irradiated cells passaged for many generations in culture. The results indicated the emergence of jackpot mutations of rapidly changing frequency, most likely reflecting the successive emergence and decline of dominant cell clones during long-term culture. These findings show that the lacZ-plasmid locus is a valid reporter for studying induced mutations in short-term cultures of both quiescent and proliferating fibroblasts. In long-term cultures, the locus is less suitable for studying induced mutations owing to the instability of the cell population.en
dc.format.extent283869 bytes-
dc.format.mimetypeapplication/pdf-
dc.language.isoenen
dc.titleMutant frequencies and spectra depend on growth state and passage number in cells cultured from transgenic lacZ-plasmid reporter mice.en
dc.typeArticleen
dc.format.digYES-
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