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The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation.

Bolin C, Boudra MT, Fernet M, Vaslin L, Pennaneach V, Zaremba T, Biard D, Cordelières FP, Favaudon V, Mégnin-Chanet F, Hall J - Cell. Mol. Life Sci. (2011)

Bottom Line: Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin.Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells.Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche, Bât. 110-112, Centre Universitaire, 91405 Orsay Cedex, France.

ABSTRACT
Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5(KD) cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5(KD) cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5(KD) cells is due to a role of Cdk5 in other pathways or the altered polymer levels.

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Quantification of poly(ADP-ribose) (PAR) formation. Representative images of PAR immunofluorescence in Control cells or Cdk5KD cells before and after exposure to 8 Gy of ionizing radiation (a, zooming set to 4) or after treatment with 1 mM H2O2 (c, zooming set to 3). Scale bar: 10 μM. b, d Quantification of the PAR staining. Data represents the mean ± SD *** (p < 10−4) based on the fluorescence intensity in over 300 cells/treatment group in 2–4 independent experiments
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Fig4: Quantification of poly(ADP-ribose) (PAR) formation. Representative images of PAR immunofluorescence in Control cells or Cdk5KD cells before and after exposure to 8 Gy of ionizing radiation (a, zooming set to 4) or after treatment with 1 mM H2O2 (c, zooming set to 3). Scale bar: 10 μM. b, d Quantification of the PAR staining. Data represents the mean ± SD *** (p < 10−4) based on the fluorescence intensity in over 300 cells/treatment group in 2–4 independent experiments

Mentions: A statistically higher basal level of PAR was detected in the Cdk5KD cells and also in the clones 1,500 and 1,501 compared to the Control cells (Fig. 4b, d and supplementary Fig. 4e). After exposure to IR, the PAR levels were increased in both cell types with significantly higher absolute levels being found in the Cdk5KD compared to the Control cells (p < 10−4, n = 300–500 cells) (Fig. 4a, b). A similar response was seen after exposure to 1 mM H2O2 (p < 10−5, n = 400–800 cells) (Fig. 4c, d). Confirmatory data obtained from clones 1,500 and 1,501 showing significantly higher absolute PAR levels compared to Control cells after treatment with IR is shown in supplementary Fig. 4e (p < 10−5, n = 200–300 cells).Fig. 4


The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation.

Bolin C, Boudra MT, Fernet M, Vaslin L, Pennaneach V, Zaremba T, Biard D, Cordelières FP, Favaudon V, Mégnin-Chanet F, Hall J - Cell. Mol. Life Sci. (2011)

Quantification of poly(ADP-ribose) (PAR) formation. Representative images of PAR immunofluorescence in Control cells or Cdk5KD cells before and after exposure to 8 Gy of ionizing radiation (a, zooming set to 4) or after treatment with 1 mM H2O2 (c, zooming set to 3). Scale bar: 10 μM. b, d Quantification of the PAR staining. Data represents the mean ± SD *** (p < 10−4) based on the fluorescence intensity in over 300 cells/treatment group in 2–4 independent experiments
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285760&req=5

Fig4: Quantification of poly(ADP-ribose) (PAR) formation. Representative images of PAR immunofluorescence in Control cells or Cdk5KD cells before and after exposure to 8 Gy of ionizing radiation (a, zooming set to 4) or after treatment with 1 mM H2O2 (c, zooming set to 3). Scale bar: 10 μM. b, d Quantification of the PAR staining. Data represents the mean ± SD *** (p < 10−4) based on the fluorescence intensity in over 300 cells/treatment group in 2–4 independent experiments
Mentions: A statistically higher basal level of PAR was detected in the Cdk5KD cells and also in the clones 1,500 and 1,501 compared to the Control cells (Fig. 4b, d and supplementary Fig. 4e). After exposure to IR, the PAR levels were increased in both cell types with significantly higher absolute levels being found in the Cdk5KD compared to the Control cells (p < 10−4, n = 300–500 cells) (Fig. 4a, b). A similar response was seen after exposure to 1 mM H2O2 (p < 10−5, n = 400–800 cells) (Fig. 4c, d). Confirmatory data obtained from clones 1,500 and 1,501 showing significantly higher absolute PAR levels compared to Control cells after treatment with IR is shown in supplementary Fig. 4e (p < 10−5, n = 200–300 cells).Fig. 4

Bottom Line: Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin.Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells.Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Centre de Recherche, Bât. 110-112, Centre Universitaire, 91405 Orsay Cedex, France.

ABSTRACT
Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5(KD) cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5(KD) cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5(KD) compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5(KD) cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5(KD) cells is due to a role of Cdk5 in other pathways or the altered polymer levels.

Show MeSH
Related in: MedlinePlus