DNA crosslinking and biological activity of a hairpin polyamide–chlorambucil conjugate

A prototype of a novel class of DNA alkylating agents, which combines the DNA crosslinking moiety chlorambucil (Chl) with a sequence-selective hairpin pyrrole-imidazole polyamide ImPy-beta-ImPy-gamma-ImPy-beta-Dp (polyamide 1), was evaluated for its ability to damage DNA and induce biological responses. Polyamide 1-Chl conjugate (1-Chl) alkylates and interstrand crosslinks DNA in cell-free systems. The alkylation occurs predominantly at 5'-AGCTGCA-3' sequence, which represents the polyamide binding site. Conjugate-induced lesions were first detected on DNA treated for 1 h with 0.1 muM 1-Chl, indicating that the conjugate is at least 100-fold more potent than Chl. Prolonged incubation allowed for DNA damage detection even at 0.01 muM concentration. Treatment with 1-Chl decreased DNA template activity in simian virus 40 (SV40) in vitro replication assays. 1-Chl inhibited mammalian cell growth, genomic DNA replication and cell cycle progression, and arrested cells in the G(2)/M phase. Moreover, cellular effects were observed at 1-Chl concentrations similar to those needed for DNA damage in cell-free systems. Neither of the parent compounds, unconjugated Chl or polyamide 1, demonstrated any cellular activity in the same concentration range. The conjugate molecule 1-Chl possesses the sequence-selectivity of a polyamide and the enhanced DNA reactivity of Chl

Activation of DNA-PK by Ionizing Radiation Is Mediated by Protein Phosphatase 6

DNA-dependent protein kinase (DNA-PK) plays a critical role in DNA damage repair, especially in non-homologous end-joining 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 PP1γ1 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

siRNA knockdown of PP6R1 or PP6c decreases repair of DNA double-strand breaks and sensitizes glioblastoma cells to radiation.

<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

siRNA knockdown of PP6R1 significantly decreases DNA-PK kinase activity.

<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

PP6R1 associates with DNA-PK in human cancer cells.

<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

siRNA knockdown of DNA-PKcs abrogates IR-induced PP6R1 nuclear localization.

<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