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Cell-cycle regulation of DNA damage-induced expression of the suppressor gene PML
1Department of Clinical Oncology, Chinese University of Hong Kong, Sir Y. K. Pao Centre for Cancer, Prince of Wales Hospital, Shatin. chanyhjohn@cujk.edu.hk
Abstract:
The promyelocytic leukemia (PML) gene, which encodes a growth- and transformation-suppressor, has been identified at the non-random chromosomal translocation break point t(15; 17)(q22; q12) of acute promyelocytic leukemia. To elucidate if PML may play a role in cellular response to DNA damage, PML expression was analyzed by immunofluorescence staining in HeLa cells treated with ionizing radiation (IR) and cisplatin. Our studies demonstrated IR at 20Gy, and cisplatin at 6 micrograms/ml caused more than 5-10 fold increases in PML protein expression in the PML Oncogenic Domain (POD) by immunofluorescent staining. Northern blotting showed that there was no gross increase in mRNA levels indicating that the induction is a post-transcriptional event. Flow cytometry showed that HeLa cells treated with IR were progressively arrested in G1, which correlates with the optimal expression of PML in the cell cycle. To determine if PML expression was under the control of the tumor suppressor p53, which is known to arrest cells in G1, HeLa cells were transfected with the wild-type p53 gene. PML expression in p53 transduced cells were 5-10 fold higher than the control, indicating that the enhanced expression of PML is apparently dependent on the p53 pathway. These data also indicate that PML may play an important role in cellular response to DNA damage such as DNA repair or apoptosis during G1 arrest.
Insights
The promyelocytic leukemia (PML) gene
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- The promyelocytic leukemia (PML) gene acts as a growth and transformation suppressor.
- PML is implicated in acute promyelocytic leukemia due to a specific chromosomal translocation t(15;17).
Purpose of the Study:
- To investigate the role of PML in cellular responses to DNA damage.
- To analyze PML gene expression following DNA damage induction.
Main Methods:
- HeLa cells were treated with ionizing radiation (IR) and cisplatin.
- PML protein expression was analyzed using immunofluorescence staining.
- mRNA levels were assessed via Northern blotting, and cell cycle progression was monitored by flow cytometry.
- The influence of the p53 tumor suppressor pathway was examined by transfecting cells with wild-type p53.
Main Results:
- Ionizing radiation (20Gy) and cisplatin (6 µg/ml) significantly increased PML protein expression (5-10 fold) post-transcriptionally.
- HeLa cells treated with IR exhibited G1 cell cycle arrest, correlating with peak PML expression.
- Enhanced PML expression was observed in p53-transduced cells, indicating p53 pathway dependence.
Conclusions:
- PML protein expression is upregulated post-transcriptionally in response to DNA damage.
- PML's enhanced expression is dependent on the p53 pathway.
- PML likely plays a crucial role in DNA repair or apoptosis during the G1 arrest phase following DNA damage.