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DNA Damage Induces p53-dependent Down-regulation of hCHK1
1Molecular Pharmacology Unit, Department of Oncology, Istituto di Ricerche Farmacologiche "Mario Negri," Via Eritrea 62, 20157 Milan, Italy. damia@irfmn.mnegri.it
Abstract:
The levels of the human checkpoint gene hCHK1 were measured in human cancer cells growing in vitro after treatment with the DNA damaging agent cis-dichlorodiammine platinum(II) (DDP). Treatment of human cancer cell lines with DDP induced a decrease in the hCHK1 protein levels starting 6 h after treatment, with a further decline at 24 and 48 h. A similar decrease in the levels of hCHK1 was found at the mRNA level by using Northern blot analysis. By using isogenic cell systems in which p53 was disrupted either by transfection with HPV-E6 or by targeted homologous recombination, we found that the DNA damage-induced down-regulation of hCHK1 was only observable in wild type p53-expressing cells, with only a minor decline in the hCHK1 levels observable 48 h after treatment in cells with disrupted p53. Similarly, treatment of mutant p53-expressing human cancer cell lines with DDP did not result in changes in the levels of hCHK1. The p53-dependent down-regulation of hCHK1 is likely to be at transcriptional levels, as suggested by the lack of down-regulation of the hCHK1 when transfected under the control of a heterologous viral promoter. In addition, p53 is able to down-regulate the luciferase activity under the control of the 5' flanking region of the hCHK1 gene. The data suggest a strict link between p53 and hCHK1 governing the activation and repression of the G(2) checkpoint in which both proteins participate.
Insights
DNA damage from cis-diamminedichloroplatinum(II) (DDP) reduces human checkpoint gene hCHK1 levels in cancer cells. This p53-dependent down-regulation occurs at the transcriptional level, impacting the G(2) checkpoint.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Cycle Regulation
Background:
- The human checkpoint gene hCHK1 plays a crucial role in cell cycle control.
- DNA-damaging agents like cis-diamminedichloroplatinum(II) (DDP) are used in cancer therapy.
- Understanding the regulation of hCHK1 is vital for cancer treatment strategies.
Purpose of the Study:
- To investigate the effect of DDP on hCHK1 levels in human cancer cells.
- To determine the role of the p53 tumor suppressor protein in the regulation of hCHK1.
- To elucidate the mechanism underlying DDP-induced hCHK1 down-regulation.
Main Methods:
- Treatment of human cancer cell lines with DDP.
- Measurement of hCHK1 protein and mRNA levels using Western blot and Northern blot analysis.
- Utilizing isogenic cell systems with disrupted or wild-type p53.
- Assessing transcriptional regulation using reporter gene assays.
Main Results:
- DDP treatment led to a significant decrease in hCHK1 protein and mRNA levels.
- This down-regulation was dependent on the presence of wild-type p53.
- Cells with disrupted or mutant p53 showed minimal changes in hCHK1 levels after DDP treatment.
- Evidence suggests p53-mediated transcriptional repression of hCHK1.
Conclusions:
- p53 strictly regulates the down-regulation of hCHK1 in response to DNA damage.
- The G(2) checkpoint activation and repression involve a coordinated interplay between p53 and hCHK1.
- These findings highlight a critical molecular pathway in cancer cells' response to chemotherapy.