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[p21(WAF1) transfection decreases sensitivity of K562 cells to VP-16]
Hui Yang1, Shen-Wu Wang, Hui-Jun Yin
1Department of Pediatrics, People's Hospital, Peking University, Beijing 100044, China.
Abstract:
The objective of this study was to explore the effect of p21(WAF1) on the sensitivity to chemotherapeutic agent VP-16, etoposide, in leukemia cell line K562. A p21(WAF1) retroviral expression vector was constructed, and mediated by FuGENE(TM)6, it was transfected into K562 cells, which without p21(WAF1) expression. The ecotropic expression of p21(WAF1) in K562 cells was identified by RT-PCR and Western blot, and named K562-p21(WAF1) cell. The K562-p21(WAF1) cells were exposed to VP-16 at different concentrations and different times, then, the sensitivity to VP-16 was examined by cell viability and MTT assay, the apoptosis induced by VP-16 was examined by DNA fragments electrophoresis and flow cytometric Annexin V-PI dual labeling technique. The results showed that the ecotropic expression of p21(WAF1) decreased the sensitivity of K562 cells to VP-16. After treatment of VP-16, the DNA ladder was examined in control K562-neo cells at 20 microgram/ml, but in K562-p21(WAF1) cells at 80 microgram/ml. With FCM, the number of apoptotic K562-neo cells was 14.9% and 25.4% respectively after treated with 20 microgram/ml VP-16 for 12 and 24 hours, and it was 6.94% and 10.96% in K562-p21(WAF1) cells. Our results suggest that the expression of p21(WAF1) could reduce the sensitivity of K562 cells to VP-16 and inhibit the apoptosis of K562 cells
Insights
The study found that increased expression of p21(WAF1) in K562 leukemia cells reduces their sensitivity to the chemotherapy drug etoposide (VP-16). This suggests p21(WAF1) inhibits etoposide-induced apoptosis in these cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Context:
- Leukemia cell lines are crucial models for studying chemotherapy resistance.
- The protein p21(WAF1) is a known cell cycle inhibitor.
- Etoposide (VP-16) is a widely used chemotherapeutic agent.
Purpose:
- To investigate the impact of p21(WAF1) expression on K562 leukemia cell sensitivity to etoposide (VP-16).
Summary:
- K562 cells were engineered to express p21(WAF1) (K562-p21(WAF1)) and compared to control cells (K562-neo).
- Sensitivity to VP-16 was assessed via cell viability, MTT assays, DNA fragmentation, and flow cytometry.
- Results demonstrated that K562-p21(WAF1) cells required higher VP-16 concentrations and showed reduced apoptosis compared to K562-neo cells.
Impact:
- The findings indicate that p21(WAF1) confers resistance to etoposide in K562 leukemia cells.
- This suggests p21(WAF1) may play a role in chemotherapy resistance mechanisms in leukemia.
- Understanding this interaction could inform strategies to overcome VP-16 resistance.
