Phosphorylated Histone 2AX Research Articles

Catalase ameliorates polychlorinated biphenyl-induced cytotoxicity in nonmalignant human breast epithelial cells

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants believed to adversely affect cellular processes. We investigated the hypothesis that PCB-induced changes in the levels of cellular reactive oxygen species (ROS) induce DNA damage resulting in cytotoxicity. Exponentially growing cultures of human nonmalignant breast epithelial cells (MCF10A) were incubated with PCBs for 3 days and assayed for cell number, ROS levels, DNA damage, and cytotoxicity. Exposure to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) or 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of 4-chlorobiphenyl (PCB3), significantly decreased cell number and MTS reduction and increased the percentage of cells with sub-G 1 DNA content. Results from electron paramagnetic resonance (EPR) spectroscopy showed a 4-fold increase in the steady-state levels of ROS, which was suppressed in cells pretreated with catalase. EPR measurements in cells treated with 4-Cl-BQ detected the presence of a semiquinone radical, suggesting that the increased levels of ROS could be due to the redox cycling of 4-Cl-BQ. A dose-dependent increase in micronuclei frequency was observed in PCB-treated cells, consistent with an increase in histone 2AX phosphorylation. Treatment of cells with catalase blunted the PCB-induced increase in micronuclei frequency and H2AX phosphorylation that was consistent with an increase in cell survival. Our results demonstrate a PCB-induced increase in cellular levels of ROS causing DNA damage, resulting in cell killing.

BRCA1 and Tip60 determine the cellular response to ultraviolet irradiation through distinct pathways

The histone acetyltransferase Tip60 regulates the apoptotic response to ultraviolet (UV) irradiation. A previously suggested mechanism for this regulation consists of the ability of Tip60 to coactivate transcription by the tumor suppressor p53. In this study, we show that Tip60 is required for the early DNA damage response (DDR) to UV, including the phosphorylation of histone 2AX, c-Jun N-terminal kinases (JNKs), and ataxia telangiectasia–related substrates. In contrast, p53 was not required for UV-induced DDR. Rather, p53 accumulation by either knockdown of Mdm2 or addition of an Mdm2 inhibitor, Nutlin-3, before irradiation strongly attenuated the UV-induced DDR and increased cell survival. This protective effect of preaccumulated p53 was mediated, at least in part, by the increased expression of CDKN1A/p21, subsequent down-regulation of BRCA1, and impaired JNK activation accompanied by decreased association of replication protein A with chromatin. We conclude that Tip60 enables UV-induced DDR signaling even in the absence of p53, whereas preaccumulated p53 suppresses UV-induced DDR by reducing the levels of BRCA1.

Enhanced Expression of RAD51 Associating Protein-1 Is Involved in the Growth of Intrahepatic Cholangiocarcinoma Cells

Intrahepatic cholangiocarcinoma (ICC) is the second most common primary cancer in the liver, and its incidence is increasing in developed countries. To discover novel molecular targets for the diagnosis and treatment of ICCs, we earlier analyzed expression profiles of 25 ICCs using a cDNA microarray containing 27,648 genes. In this study, we focused on the RAD51 associating protein-1 (RAD51AP1) gene because its expression was frequently elevated in our microarray data. Quantitative PCR confirmed that RAD51AP1 expression was elevated in the great majority of the ICCs examined. Immunohistochemical analysis with anti-RAD51AP1 antibody further corroborated its accumulation in 14 of 23 ICC tissues (61%). Notably, suppression of RAD51AP1 by short interfering RNA resulted in growth suppression of cholangiocarcinoma cells, suggesting its involvement in the development and/or progression of ICC. Because RAD51AP1 interacts with RAD51, a molecule involved in DNA repair, we investigated whether RAD51AP1 is implicated in DNA strand breaks using gamma-irradiation. As a result, gamma-irradiation augmented RAD51AP1 protein expression and brought a focus formation in the nuclei, where accumulated RAD51AP1 colocalized with phosphorylated histone 2AX (gamma-H2AX) and RAD51. These data suggest that RAD51AP1 may play a role in cell proliferation as well as DNA repair. Our findings may contribute to the better understanding of cholangiocarcinogenesis and open a new avenue to the development of novel therapeutic and/or diagnostic approach to this type of tumor.

BCL6 Attenuates DNA Damage Sensing in Normal and Malignant B-Cells by Directly Repressing ATR.

The BCL6 (B-Cell-Lymphoma-6) transcriptional repressor is a critical oncogene in B-cell lymphomas and is required for establishment of germinal centers by normal B-cells. However, the mechanisms by which BCL6 licenses germinal center formation and lymphomagenesis are unknown. To characterize this mechanism we identified BCL6 target genes by expression arrays and high throughput chromatin immunoprecipitations. Remarkably, a number of these target genes were critical mediators of DNA damage sensing checkpoints including ATR and p53. Therefore, we hypothesized that BCL6 could attenuate DNA damage sensing by silencing these genes, which is likely a critical attribute for survival and proliferation of germinal center B-cells cells undergoing somatic hypermutation (SHM) and class switch recombination (CSR). Accordingly, we found that expression of BCL6 in normal diploid fibroblasts could block cellular sensing of DNA damage as demonstrated by loss of histone 2AX (H2AX) phosphorylation and delayed repair of double strand breaks. Repression of ATR (but not p53 or other targets) was required for this phenotype. This is a physiological effect since the same result was observed when BCL6 was expressed in purified primary human tonsilar mature B-cells. Reciprocally, shRNA knockdown of BCL6 in B-cell lymphomas rescued repression of ATR, enhanced H2AX phosphorylation and accelerated repair of double strand breaks, independent of the status of p53. shRNA knockdown of BCL6 caused a marked increase of apoptosis in lymphoma cells in response to DNA damage, due to restored DNA damage checkpoint functions. Importantly, BCL6 knockdown had an identical effect on ATR levels, H2AX phosphorylation, DNA damage, and survival in purified primary human germinal center centroblasts. These results suggest that a major role of BCL6 in germinal center formation is to attenuate cellular response to DNA damage occurring as a byproduct of CSR and SHM. The same mechanism also seems to be required for lymphomagenesis, since we observed that sustained BCL6 expression in human primary mature B-cells leads to aberrant survival properties and genomic instability. Moreover, BCL6 blockade using a specific inhibitor molecule designed by our lab induces apoptosis in lymphoma cells and synergizes with DNA damaging agents. Therefore, we have identified a critical mechanism of action of the BCL6 oncoprotein in normal and pathogenic states and show that specific targeting of BCL6 could synergize with chemotherapy drugs for the therapy of B-cell lymphomas.

Silymarin and silibinin cause G1 and G2–M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin

Here, we assessed and compared the anticancer efficacy and associated mechanisms of silymarin and silibinin in human prostate cancer (PCA) PC3 cells; silymarin is comprised of silibinin and its other stereoisomers, including isosilybin A, isosilybin B, silydianin, silychristin and isosilychristin. Silymarin and silibinin (50-100 microg/ml) inhibited cell proliferation, induced cell death, and caused G1 and G2-M cell cycle arrest in a dose/time-dependent manner. Molecular studies showed that G1 arrest was associated with a decrease in cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)4, CDK6 and CDK2 protein levels, and CDK2 and CDK4 kinase activity, together with an increase in CDK inhibitors (CDKIs) Kip1/p27 and Cip1/p21. Further, both agents caused cytoplasmic sequestration of cyclin D1 and CDK2, contributing to G1 arrest. The G2-M arrest by silibinin and silymarin was associated with decreased levels of cyclin B1, cyclin A, pCdc2 (Tyr15), Cdc2, and an inhibition of Cdc2 kinase activity. Both agents also decreased the levels of Cdc25B and cell division cycle 25C (Cdc25C) phosphatases with an increased phosphorylation of Cdc25C at Ser216 and its translocation from nucleus to the cytoplasm, which was accompanied by an increased binding with 14-3-3beta. Both agents also increased checkpoint kinase (Chk)2 phosphorylation at Thr68 and Ser19 sites, which is known to phosphorylate Cdc25C at Ser216 site. Chk2-specific small interfering RNA largely attenuated the silymarin and silibinin-induced G2-M arrest. An increase in the phosphorylation of histone 2AX and ataxia telangiectasia mutated was also observed. These findings indicate that silymarin and silibinin modulate G1 phase cyclins-CDKs-CDKIs for G1 arrest, and the Chk2-Cdc25C-Cdc2/cyclin B1 pathway for G2-M arrest, together with an altered subcellular localization of critical cell cycle regulators. Overall, we observed comparable effects for both silymarin and silibinin at equal concentrations by weight, suggesting that silibinin could be a major cell cycle-inhibitory component in silymarin. However, other silibinin stereoisomers present in silymarin also contribute to its efficacy, and could be of interest for future investigation.

Single-stranded DNA Induces Ataxia Telangiectasia Mutant (ATM)/p53-dependent DNA Damage and Apoptotic Signals

Single-stranded DNA has been speculated to be the initial signal in the DNA damage signaling pathway. We showed that introduction of single-stranded DNA with diverse sequences into mammalian cells induced DNA damage as well as apoptosis signals. Like DNA damaging agents, single-stranded DNA up-regulated p53 and activated the nuclear kinase ataxia telangiectasia mutant (ATM) as evidenced by phosphorylation of histone 2AX, an endogenous ATM substrate. Single-stranded DNA also triggered apoptosis as evidenced by the formation of caspase-dependent chromosomal DNA strand breaks, cytochrome c release, and increase in reactive oxygen species production. Moreover, single-stranded DNA-induced apoptosis was reduced significantly in p53 null cells and in cells treated with ATM small interfering RNA. These results suggest that single-stranded DNA may act upstream of ATM/p53 in DNA damage signaling.