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Ubiquitous, heritable damage in cell populations that survive treatment with methotrexate
1Department of Molecular and Cell Biology and Virus Laboratory, 229 Stanley Hall, University of California, Berkeley, CA 94720-3206, USA.
Abstract:
A permanent line of mouse embryo fibroblasts was treated with concentrations of the anticancer drug methotrexate (MTX) that left 20-50% surviving colonies. The surviving population initially multiplied at a much slower rate than controls after subculture in the absence of the drug, and required 9-12 days of serial subculture, with selective growth of the faster growing cells, to approximate the control rate. To determine the distribution of growth rates of cells in the original posttreatment populations, many single cells were isolated in multiwell plates immediately after the treatment period, and the resulting clones were serially subcultured. Most of the control clones underwent about 2 population doublings per day (PD/D). Almost all the survivors of MTX treatment multiplied at heterogeneously reduced rates, ranging from 0.6 PD/D to as high as control rates for a very few clones. They maintained the reduced rates through many subcultivations. The heritability of the reduced growth rates indicates that most cells that retain proliferative capacity after treatment with MTX carry random genetic damage that is perpetuated through many divisions of their progeny. Similar results have been described for cells that survive x-irradiation, and suggest random genetic damage is a common occurrence among cells in rapidly growing tissues that survive cytotoxic treatment. It also occurs in serial subcultures of cells that had been held under the constraint of confluence for extended periods, which suggests that the accumulation of random genetic damage to somatic cells during aging of mammals underlies the reduction of growth rate and function of the cells that characterizes the aging process.
Insights
Anticancer drug methotrexate (MTX) treatment causes lasting, heritable damage to surviving mouse cells, reducing their growth rates. This genetic damage impacts cell proliferation and may contribute to aging processes.
Area of Science:
- Cell Biology
- Genetics
- Pharmacology
Background:
- Anticancer drugs like methotrexate (MTX) are used to treat cancer by targeting rapidly dividing cells.
- Understanding the long-term effects of cytotoxic treatments on surviving cells is crucial for assessing treatment efficacy and potential side effects.
Purpose of the Study:
- To investigate the growth rate distribution and long-term proliferative capacity of mouse embryo fibroblasts surviving methotrexate treatment.
- To determine if reduced growth rates in surviving cells are heritable and indicative of genetic damage.
Main Methods:
- Mouse embryo fibroblasts were exposed to methotrexate (MTX) at concentrations yielding 20-50% survival.
- Surviving single cells were isolated and their clonal populations serially subcultured to assess growth rates (population doublings per day).
- Growth rates were compared between control and MTX-treated cell populations immediately post-treatment and after extended subculture.
Main Results:
- Control cells exhibited approximately 2 population doublings per day.
- Most MTX survivors displayed heterogeneous, significantly reduced growth rates (0.6-2 PD/D) that were maintained through serial subculture.
- A few clones approached control growth rates, but the majority showed persistent, heritable reductions in proliferation.
Conclusions:
- Methotrexate treatment induces random, heritable genetic damage in surviving mouse fibroblasts, manifesting as reduced and persistent growth rates.
- This damage and its effect on cell proliferation mirror findings in cells surviving other cytotoxic treatments like X-irradiation.
- The findings suggest that accumulated random genetic damage in somatic cells may underlie age-related decline in cellular growth and function.