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Papers by Elzbieta Bolesta

Purpose: We have previously reported that many types of tumors can induce changes in human T cell... more Purpose: We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways.Experimental Design: We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection.Results: We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AK...

<p>M059K cells were transfected with control, non-targeting siRNA or specific siRNAs target... more <p>M059K cells were transfected with control, non-targeting siRNA or specific siRNAs targeted to PP5, PP1γ1, PP6c, PP6R3 and PP6R1. A. Forty-eight hours after transfection, the M059K cells were irradiated with 5 Gy or sham-irradiated. Thirty minutes after irradiation, cells were harvested and fractionated. DNA-PKcs was immunoprecipitated from nuclear fractions, and the activity of DNA-PKcs was measured by incorporation of ³²P into a DNA-PKcs-specific P53-derived peptide substrate. DNA-PKcs-deficient M059J cells were used as a negative control. The numbers were normalized to non-irradiated M059K cells and the data show the mean of counts per minute per µg protein in the eluted solution ± SD (n = 3). B. M059K cells transfected with anti-PP6c or anti-PP6R1 siRNA as described in panel A were treated with 1 µM CPT for 4 hours. RPA2 was detected in whole cell lysates by Western blot. . The slower migrating band represents the phosphorylated form of RPA2. M059J cells were used as a negative control.</p

<p>A. Two days after transfection with siRNA cells were irradiated with 10 Gy and either ha... more <p>A. Two days after transfection with siRNA cells were irradiated with 10 Gy and either harvested immediately (10 Gy) or allowed to repair DNA damage for 3 hours (10 Gy+3 hours) prior to harvesting cells. Harvested cells were embedded in agarose plugs and subjected to PFGE as described. The bars represent the fraction of DNA released from agarose plugs during PFGE, normalized to sham-irradiated control (±SEM). The data are from two independent experiments. M059J cells were used as a negative control. B. M059K cells were transfected with control siRNA or specific siRNAs targeted to DNA-PKcs, PP6R1, PP6c, PP6R3, or ARS-A. Two days after transfection, the M059K cells were irradiated with 0, 2.5, 5 Gy, replated, cultured for two weeks and scored for surviving colonies. The data points show the mean of surviving fraction ± SD (n = 3). M059J cells were used as a negative control. C. Representative Western blot for siRNA knockdown efficiency. The blot shows the level of the proteins of interest after transfection with siRNA. Ku86 is a marker for the nuclear fraction and was used as a loading control.</p

<p>A. Cell extracts from irradiated DNA-PK proficient (M059K), and deficient glioblastoma c... more <p>A. Cell extracts from irradiated DNA-PK proficient (M059K), and deficient glioblastoma cells (M059J) were immunoprecipitated with monoclonal α-DNA-PKcs antibody. Following SDS-PAGE, DNA-PKcs, PP6R1 and PP6c proteins were detected by immunostaining using specific antibodies or pre-immune serum. B. M059K cells were irradiated with 5 or 10 Gy, or sham-treated (0 Gy). One hour after radiation, the cells were lysed and cytoplasmic and nuclei were prepared. Nuclei were subjected to immunoprecipitation with DNA-PKcs antisera. C. Immunoblots of the nuclear fractions were quantified by densitometry. Standard error bars represent the mean of three independent experiments (± SD). The statistical significance of the differences between the amount of PP6c or PP6R1 in the nuclear fraction from irradiated cells, and in the nuclear fraction from non-irradiated control cells was (***, p<0.001) by Student T test.</p

<p>A. M059K cells were transfected with control siRNA or anti-DNA-PKcs siRNA, and 48 hours ... more <p>A. M059K cells were transfected with control siRNA or anti-DNA-PKcs siRNA, and 48 hours later subjected to 5 Gy. One hour post-irradiation, the cells were fixed and immunostained with anti-DNA-PKcs and PP6R1 antibodies. Upper row: orange PP6R1 and lower row: merged Dapi ,DNA-PK and PP6R1. B. M059K cells were transfected with control siRNA or anti-DNA-PKcs siRNA and subjected to irradiation 48 hours post –transfection. One hour post-irradiation the cells were fractionated into cytoplasmic and nuclear fractions. The protein levels of DNA-PKcs, PP6R1, and Ku were detected by immunoblotting.</p

Molecular and Cellular Biology, 2012

Although senescence in oncogenesis has been widely studied, little is known regarding the role of... more Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53 + cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53 − cells restored CIS to similar levels as p53 + cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth reg...

International Journal of Molecular Medicine, 2007

Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualita... more Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.

PLoS ONE, 2009

DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in n... more DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous endjoining repair of double-strand breaks such as those formed by ionizing radiation (IR) in the course of radiation therapy. Regulation of DNA-PK involves multisite phosphorylation but this is incompletely understood and little is known about protein phosphatases relative to DNA-PK. Mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation. Knockdown of other phosphatases PP5 or PP1c1 and subunits PP6R3 or ARS-A did not reduce IR activation of DNA-PK, demonstrating specificity for PP6R1. Finally, siRNA knockdown of PP6R1 or PP6 but not other phosphatases increased the sensitivity of glioblastoma cells to radiation-induced cell death to a level similar to DNA-PK deficient cells. Our data demonstrate that PP6 associates with and activates DNA-PK in response to ionizing radiation. Therefore, the PP6/PP6R1 phosphatase is a potential molecular target for radiation sensitization by chemical inhibition.

Plant Science, 2000

In this work we studied the changes in the level of abscisic acid (ABA) in the somatic embryos (S... more In this work we studied the changes in the level of abscisic acid (ABA) in the somatic embryos (SE) and in the diploid and triploid zygotic embryos (ZE) of the same cucumber line during embryogenesis and seed maturation. Different stages of seed development were selected according to days after pollination (DAP): 21, 24, 28, 35, 42 and 42 plus 14 days of storage for diploid ZE and 35 and 42 plus 14 days of storage for triploid ZE. SE were collected at five growth stages from globular to late cotyledonary. Quantitative analysis of ABA was performed using an enzyme linked immunosorbent assay (ELISA) test. Both types of embryos-somatic and zygotic-essentially differed in their levels of ABA, always being the highest for 2n ZE and lowest for SE. Although the concentration of ABA in ZE of the triploid line was higher when compared with the same DAP, when the comparison was based on embryo development, both the concentration and content of ABA was higher in the diploid line. The pattern of developmental changes in the level of ABA in the diploid ZE was consistent with that known for other species. An increase was observed during embryo development with a peak (51.1 mg g − 1 FW or 0.95 mg per embryo) at the final stage of embryo formation between 21 and 24 DAP. A sharp decrease in the ABA level then took place (more than 3-fold within 4 days) and was followed by a further reduction as the seed matured. The maximal and minimal values for ABA concentration differed about 35-fold. SE differed substantially from their zygotic counterparts not only in that the concentration of ABA was extremely low (0.005-0.011 mg g − 1 FW) but also that no significant changes occurred during embryo development and no peak of ABA concentration was observed. Other tissues of the ovule and ovary also contained ABA and could be a source of ABA for the embryo.

Clinical Cancer Research, 2011

Purpose: We have previously reported that many types of tumors can induce changes in human T cell... more Purpose: We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways.Experimental Design: We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection.Results: We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AK...

Molecular Cancer Therapeutics, 2009

In response to ionizing radiation, p53 plays a critical role in regulating DNA repair and apoptos... more In response to ionizing radiation, p53 plays a critical role in regulating DNA repair and apoptosis. Among multiple phosphorylation sites, evidence suggests that Ser46 promotes apoptotic cell death through mitochondrial outer membrane permeabilization (MOMP) and subsequent activation of the caspase 7–PARP pathway. Therefore, we investigated which phosphatase regulates Ser46 after ionizing radiation, reasoning that the responsible phosphatase should be a target for radiosensitization. We determined that both inhibition of PP2A by the cell-permeable inhibitor calyculin A and knockdown of PP2A by RNAi (a) enhanced Ser46 phosphorylation in p53 and (b) induced coincident caspase 7 and PARP cleavage in response to ionizing radiation. Furthermore, mutation of p53 Ser46 to Ala attenuated ionizing radiation–induced apoptotic signaling. Consequently, we concluded that PP2A regulates ionizing radiation–induced apoptotic signaling through dephosphorylation of p53 Ser46. [Mol Cancer Ther 2009;8(...

Purpose: We have previously reported that many types of tumors can induce changes in human T cell... more Purpose: We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways.Experimental Design: We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection.Results: We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AK...

<p>M059K cells were transfected with control, non-targeting siRNA or specific siRNAs target... more <p>M059K cells were transfected with control, non-targeting siRNA or specific siRNAs targeted to PP5, PP1γ1, PP6c, PP6R3 and PP6R1. A. Forty-eight hours after transfection, the M059K cells were irradiated with 5 Gy or sham-irradiated. Thirty minutes after irradiation, cells were harvested and fractionated. DNA-PKcs was immunoprecipitated from nuclear fractions, and the activity of DNA-PKcs was measured by incorporation of ³²P into a DNA-PKcs-specific P53-derived peptide substrate. DNA-PKcs-deficient M059J cells were used as a negative control. The numbers were normalized to non-irradiated M059K cells and the data show the mean of counts per minute per µg protein in the eluted solution ± SD (n = 3). B. M059K cells transfected with anti-PP6c or anti-PP6R1 siRNA as described in panel A were treated with 1 µM CPT for 4 hours. RPA2 was detected in whole cell lysates by Western blot. . The slower migrating band represents the phosphorylated form of RPA2. M059J cells were used as a negative control.</p

<p>A. Two days after transfection with siRNA cells were irradiated with 10 Gy and either ha... more <p>A. Two days after transfection with siRNA cells were irradiated with 10 Gy and either harvested immediately (10 Gy) or allowed to repair DNA damage for 3 hours (10 Gy+3 hours) prior to harvesting cells. Harvested cells were embedded in agarose plugs and subjected to PFGE as described. The bars represent the fraction of DNA released from agarose plugs during PFGE, normalized to sham-irradiated control (±SEM). The data are from two independent experiments. M059J cells were used as a negative control. B. M059K cells were transfected with control siRNA or specific siRNAs targeted to DNA-PKcs, PP6R1, PP6c, PP6R3, or ARS-A. Two days after transfection, the M059K cells were irradiated with 0, 2.5, 5 Gy, replated, cultured for two weeks and scored for surviving colonies. The data points show the mean of surviving fraction ± SD (n = 3). M059J cells were used as a negative control. C. Representative Western blot for siRNA knockdown efficiency. The blot shows the level of the proteins of interest after transfection with siRNA. Ku86 is a marker for the nuclear fraction and was used as a loading control.</p

<p>A. Cell extracts from irradiated DNA-PK proficient (M059K), and deficient glioblastoma c... more <p>A. Cell extracts from irradiated DNA-PK proficient (M059K), and deficient glioblastoma cells (M059J) were immunoprecipitated with monoclonal α-DNA-PKcs antibody. Following SDS-PAGE, DNA-PKcs, PP6R1 and PP6c proteins were detected by immunostaining using specific antibodies or pre-immune serum. B. M059K cells were irradiated with 5 or 10 Gy, or sham-treated (0 Gy). One hour after radiation, the cells were lysed and cytoplasmic and nuclei were prepared. Nuclei were subjected to immunoprecipitation with DNA-PKcs antisera. C. Immunoblots of the nuclear fractions were quantified by densitometry. Standard error bars represent the mean of three independent experiments (± SD). The statistical significance of the differences between the amount of PP6c or PP6R1 in the nuclear fraction from irradiated cells, and in the nuclear fraction from non-irradiated control cells was (***, p<0.001) by Student T test.</p

<p>A. M059K cells were transfected with control siRNA or anti-DNA-PKcs siRNA, and 48 hours ... more <p>A. M059K cells were transfected with control siRNA or anti-DNA-PKcs siRNA, and 48 hours later subjected to 5 Gy. One hour post-irradiation, the cells were fixed and immunostained with anti-DNA-PKcs and PP6R1 antibodies. Upper row: orange PP6R1 and lower row: merged Dapi ,DNA-PK and PP6R1. B. M059K cells were transfected with control siRNA or anti-DNA-PKcs siRNA and subjected to irradiation 48 hours post –transfection. One hour post-irradiation the cells were fractionated into cytoplasmic and nuclear fractions. The protein levels of DNA-PKcs, PP6R1, and Ku were detected by immunoblotting.</p

Molecular and Cellular Biology, 2012

Although senescence in oncogenesis has been widely studied, little is known regarding the role of... more Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53 + cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53 − cells restored CIS to similar levels as p53 + cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth reg...

International Journal of Molecular Medicine, 2007

Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualita... more Aberrant glycosylation is a universal feature of cancer cells. There are quantitative and qualitative changes in expression of gangliosides observed in tumors of a neuroectodermal origin such as neuroblastoma, melanoma and astrocytoma. The presence of large amounts of GD2 ganglioside on neuroblastoma cells, as compared to normal cells, opens the possibilities to use the tumor-associated carbohydrate antigen in diagnosis and immunotherapeutic approaches. In the quest for immunogens potentially capable of eliciting anti-GD2 ganglioside immune responses, we performed affinity purification of phage-displayed peptides from the LX-8 library (12-mer containing disulphide bridge). The library was screened with the biotinylated anti-GD2 ganglioside 14G2a mAb monoclonal antibody. Our goal was to isolate and characterize peptide mimics of GD2 ganglioside. Numerous individual phage clones that bound 14G2a mAb were identified with the application of immunoblotting technique in the phage pools yielded from the pannings. The phage-borne peptides were tested for their anti-GD2 ganglioside antibody binding ability using ELISA. Among these clones five different phage-displayed peptide sequences were identified. Moreover, we showed that the secondary structure of the peptides, stabilized by the disulfide bridging between cysteine residues at positions 2 and 11, was crucial for the binding of the peptides to 14G2a mAb. In a separate set of experiments, we observed a competition of the peptides, expressed on phages as well as in their synthetic form, with the nominal antigen GD2 ganglioside expressed on IMR-32 neuroblastoma cells for binding to 14G2a mAb. Based on the obtained results we concluded that all of these 5 peptides were mimics of the GD2 ganglioside.

PLoS ONE, 2009

DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in n... more DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous endjoining repair of double-strand breaks such as those formed by ionizing radiation (IR) in the course of radiation therapy. Regulation of DNA-PK involves multisite phosphorylation but this is incompletely understood and little is known about protein phosphatases relative to DNA-PK. Mass spectrometry analysis revealed that DNA-PK interacts with the protein phosphatase-6 (PP6) SAPS subunit PP6R1. PP6 is a heterotrimeric enzyme that consists of a catalytic subunit, plus one of three PP6 SAPS regulatory subunits and one of three ankyrin repeat subunits. Endogenous PP6R1 co-immunoprecipitated DNA-PK, and IR enhanced the amount of complex and promoted its import into the nucleus. In addition, siRNA knockdown of either PP6R1 or PP6 significantly decreased IR activation of DNA-PK, suggesting that PP6 activates DNA-PK by association and dephosphorylation. Knockdown of other phosphatases PP5 or PP1c1 and subunits PP6R3 or ARS-A did not reduce IR activation of DNA-PK, demonstrating specificity for PP6R1. Finally, siRNA knockdown of PP6R1 or PP6 but not other phosphatases increased the sensitivity of glioblastoma cells to radiation-induced cell death to a level similar to DNA-PK deficient cells. Our data demonstrate that PP6 associates with and activates DNA-PK in response to ionizing radiation. Therefore, the PP6/PP6R1 phosphatase is a potential molecular target for radiation sensitization by chemical inhibition.

Plant Science, 2000

In this work we studied the changes in the level of abscisic acid (ABA) in the somatic embryos (S... more In this work we studied the changes in the level of abscisic acid (ABA) in the somatic embryos (SE) and in the diploid and triploid zygotic embryos (ZE) of the same cucumber line during embryogenesis and seed maturation. Different stages of seed development were selected according to days after pollination (DAP): 21, 24, 28, 35, 42 and 42 plus 14 days of storage for diploid ZE and 35 and 42 plus 14 days of storage for triploid ZE. SE were collected at five growth stages from globular to late cotyledonary. Quantitative analysis of ABA was performed using an enzyme linked immunosorbent assay (ELISA) test. Both types of embryos-somatic and zygotic-essentially differed in their levels of ABA, always being the highest for 2n ZE and lowest for SE. Although the concentration of ABA in ZE of the triploid line was higher when compared with the same DAP, when the comparison was based on embryo development, both the concentration and content of ABA was higher in the diploid line. The pattern of developmental changes in the level of ABA in the diploid ZE was consistent with that known for other species. An increase was observed during embryo development with a peak (51.1 mg g − 1 FW or 0.95 mg per embryo) at the final stage of embryo formation between 21 and 24 DAP. A sharp decrease in the ABA level then took place (more than 3-fold within 4 days) and was followed by a further reduction as the seed matured. The maximal and minimal values for ABA concentration differed about 35-fold. SE differed substantially from their zygotic counterparts not only in that the concentration of ABA was extremely low (0.005-0.011 mg g − 1 FW) but also that no significant changes occurred during embryo development and no peak of ABA concentration was observed. Other tissues of the ovule and ovary also contained ABA and could be a source of ABA for the embryo.

Clinical Cancer Research, 2011

Purpose: We have previously reported that many types of tumors can induce changes in human T cell... more Purpose: We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways.Experimental Design: We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection.Results: We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AK...

Molecular Cancer Therapeutics, 2009

In response to ionizing radiation, p53 plays a critical role in regulating DNA repair and apoptos... more In response to ionizing radiation, p53 plays a critical role in regulating DNA repair and apoptosis. Among multiple phosphorylation sites, evidence suggests that Ser46 promotes apoptotic cell death through mitochondrial outer membrane permeabilization (MOMP) and subsequent activation of the caspase 7–PARP pathway. Therefore, we investigated which phosphatase regulates Ser46 after ionizing radiation, reasoning that the responsible phosphatase should be a target for radiosensitization. We determined that both inhibition of PP2A by the cell-permeable inhibitor calyculin A and knockdown of PP2A by RNAi (a) enhanced Ser46 phosphorylation in p53 and (b) induced coincident caspase 7 and PARP cleavage in response to ionizing radiation. Furthermore, mutation of p53 Ser46 to Ala attenuated ionizing radiation–induced apoptotic signaling. Consequently, we concluded that PP2A regulates ionizing radiation–induced apoptotic signaling through dephosphorylation of p53 Ser46. [Mol Cancer Ther 2009;8(...