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Regulation of p53 stabilization by DNA damage and protein kinase C
Cassie L Johnson1, Dongmei Lu, Jie Huang
1Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Institute for Cancer Research, Fort Worth, Texas 76107, USA.
Abstract:
We have demonstrated previously that the protein kinase C (PKC) signal transduction pathway acts upstream of caspases to regulate caspase activation and apoptosis induced by the DNA-damaging agent cisplatin (CP). In the present study, we have examined whether PKC influences p53 and, hence, cellular sensitivity/resistance to CP. The basal p53 level was low in HeLa cells but was elevated in CP-resistant HeLa (HeLa/CP) cells. CP had no effect on the p53 content in HeLa cells, but it caused p53 accumulation in HeLa/CP cells. Rottlerin, a PKCdelta inhibitor that prevents CP-induced proteolytic activation of PKCdelta, caused an accumulation of p53 in HeLa cells when treated in conjunction with CP, but it had no additional effect in HeLa/CP cells. The ability of rottlerin to prevent proteolytic activation of PKCdelta or to induce accumulation of p53 by CP was compromised in HeLa/CP cells. PKC activator phorbol 12, 13-dibutyrate attenuated constitutive p53 levels in both HeLa and HeLa/CP cells. Whereas the combination of rottlerin and CP increased the half-life of p53 in HeLa cells, CP alone was sufficient to stabilize p53 in HeLa/CP cells. These results suggest that both DNA damage and inhibition of proteolytic activation of PKCdelta by CP were necessary for the stabilization of p53 in HeLa cells. Furthermore, an increase in p53 was not associated with enhanced sensitivity of HeLa cells to CP.
Insights
Protein Kinase C (PKC) signaling influences p53 levels and cellular response to cisplatin (CP) DNA damage. Inhibiting PKCdelta with rottlerin stabilizes p53 in HeLa cells, but this effect is altered in cisplatin-resistant cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The protein kinase C (PKC) pathway regulates apoptosis induced by DNA-damaging agents like cisplatin (CP).
- The role of PKC in modulating p53 levels and cellular sensitivity to CP remains to be fully elucidated.
Purpose of the Study:
- To investigate the influence of PKC on p53 expression and cellular sensitivity/resistance to cisplatin.
- To determine how PKC inhibition affects p53 stabilization and cellular response to DNA damage.
Main Methods:
- Utilized HeLa and cisplatin-resistant HeLa (HeLa/CP) cell lines.
- Administered cisplatin (CP), rottlerin (PKCdelta inhibitor), and phorbol 12, 13-dibutyrate (PKC activator).
- Assessed p53 levels, p53 half-life, and PKCdelta activation through Western blotting and drug treatments.
Main Results:
- Basal p53 levels were higher in HeLa/CP cells compared to HeLa cells.
- Cisplatin induced p53 accumulation in HeLa/CP cells but not in HeLa cells.
- Rottlerin combined with CP caused p53 accumulation in HeLa cells, an effect compromised in HeLa/CP cells.
- PKC activation by phorbol 12, 13-dibutyrate reduced p53 levels in both cell types.
- CP alone stabilized p53 in HeLa/CP cells, while rottlerin and CP together stabilized p53 in HeLa cells.
Conclusions:
- PKC signaling, particularly PKCdelta, plays a significant role in regulating p53 stabilization in response to cisplatin-induced DNA damage.
- Cisplatin resistance in HeLa/CP cells is associated with altered p53 regulation and compromised PKCdelta activation.
- Increased p53 levels did not correlate with enhanced cisplatin sensitivity in HeLa cells, suggesting complex regulatory mechanisms.