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Attenuation of G2 checkpoint function precedes human cell immortalization
W K Kaufmann1, E N Levedakou, H L Grady
1Department of Pathology, University of North Carolina at Chapel Hill 27599-7295.
Abstract:
We have investigated the hypothesis that attenuation of the G2 checkpoint, which delays entry into mitosis in response to damage to DNA and protects against clastogenesis, may contribute to the genetic instability of immortal human cell lines. IMR-90 normal human fibroblasts displayed stringent G2 checkpoint response to gamma-radiation-induced DNA damage. Irradiation with 1.5 Gy induced 98% inhibition of mitosis and 79% inhibition of cyclin B1/p34CDC2 kinase activity within 2 h. SV40-transformed IMR-90 cells with extended in vitro proliferative lifespan and immortal derivative cells displayed significantly less radiation-induced G2 delay (60-70%) and less inhibition of cyclin B1/p34CDC2 protein kinase activity (43-46%) than was seen in normal cells. Two other SV40-transformed lines and a fibrosarcoma line displayed a similar attenuation of G2 checkpoint function. The attenuation of G2 checkpoint function in SV40 transformed IMR-90 cells was associated with elevated levels of expression of cyclin B1 (8-fold greater) and p34CDC2 (2.5-fold greater). By allowing cells with damaged chromatids to enter mitosis, an attenuation of G2 checkpoint function in finite lifespan cells may promote the genetic alterations necessary for the conversion to immortality.
Insights
Immortal human cells show a weakened G2 checkpoint, allowing damaged DNA to enter mitosis. This genetic instability may be key to cells becoming immortal.
Area of Science:
- Cell biology
- Genetics
- Cancer research
Background:
- The G2 checkpoint prevents mitosis in cells with DNA damage, protecting against genetic instability.
- Immortal human cell lines often exhibit genetic instability, but the underlying mechanisms are not fully understood.
Purpose of the Study:
- To investigate if a weakened G2 checkpoint contributes to the genetic instability observed in immortal human cell lines.
- To determine if attenuation of the G2 checkpoint is a factor in the conversion of normal cells to an immortal state.
Main Methods:
- Comparing the G2 checkpoint response to gamma-radiation-induced DNA damage in normal human fibroblasts (IMR-90) versus SV40-transformed and immortal derivative cells.
- Quantifying the inhibition of mitosis and cyclin B1/p34CDC2 kinase activity post-irradiation.
- Measuring the expression levels of cyclin B1 and p34CDC2 in different cell types.
Main Results:
- Normal IMR-90 fibroblasts exhibited a stringent G2 checkpoint response to gamma radiation.
- SV40-transformed and immortal cells showed significantly attenuated G2 delay and reduced inhibition of cyclin B1/p34CDC2 kinase activity.
- Elevated expression of cyclin B1 (8-fold) and p34CDC2 (2.5-fold) was observed in SV40-transformed IMR-90 cells.
Conclusions:
- Attenuation of the G2 checkpoint is a characteristic of immortal human cell lines.
- This weakened checkpoint allows cells with damaged chromatids to enter mitosis, potentially promoting genetic alterations necessary for immortality.
- The findings suggest a mechanism by which normal cells may acquire immortality through genetic instability driven by checkpoint defects.