Identification and fate of a marker chromosome in methotrexate-resistant V79,B7 cells by flow karyotyping and sorting, metaphase analysis and in situ hybridization.
The chromosomes from a methotrexate (MTX)-resistant and its parental V79,B7 Chinese hamster cell line were analysed by the combined use of flow karyotyping and sorting, metaphase analysis and in situ hybridization with a probe for the dihydrofolate reductase (DHFR) gene responsible for methotrexate resistance. A marker chromosome with an elongated arm carrying the amplified DHFR gene was identified by in situ hybridization of metaphase cells of the methotrexate-resistant line. In the flow karyotype the marker chromosome was found as an additional peak with a higher DNA content compared with the largest chromosome of the sensitive line. This was additionally verified by G-banding of the chromosomes sorted from the marker peak. Several other chromosomal rearrangements not associated with the amplified gene could be identified in the methotrexate-resistant line by the combined use of flow karyotyping and metaphase analysis. The fate of the original marker chromosome was studied in cells growing several weeks in the absence of methotrexate, comparing flow karyotyping and metaphase analysis. The original marker chromosome was lost in about 50% of the cells after 5 weeks and in about 60% of the cells after 8 weeks; between 80 and 90% of the cells, however, contained marker chromosomes of various sizes. The MTX-resistance decreased in parallel during loss of the original marker chromosome. In conclusion, the study shows that the power of cytogenetic analysis is improved by the combined use of conventional cytogenetics, molecular cytogenetics and flow cytometry.
Nüsse
M
M
GSF-Institut für Biophysikalische Strahlenforschung, Neuherberg, Germany.
Viaggi
S
S
Bonatti
S
S
D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D002872:Chromosome Deletion; D004247:DNA / Q000032:analysis*; D004351:Drug Resistance / Q000235:genetics; D005453:Fluorescence; D005819:Genetic Markers / Q000235:genetics; D017403:In Situ Hybridization; D007621:Karyotyping / Q000379:methods; D008677:Metaphase / Q000235:genetics*; D008727:Methotrexate / Q000494:pharmacology*
D005819:Genetic Markers; D004247:DNA; D008727:Methotrexate