Repression of apurinic/apyrimidinic endonuclease by p53-dependent apoptosis in hydronephrosis-induced rat kidney

p53 plays a major role in apoptosis through activation of pro-apoptotic gene Bax. It also regulates apurinic/apyrimidinic endonuclease (APE) expression in the base excision repair pathway against oxidative DNA damages. This study investigated whether p53-dependent apoptosis is correlated with APE using an experimental rat model of hydronephrosis. Hydronephrosis was induced by partial ligation of the right ureter. Animals were sacrificed on scheduled time after unilateral ureteral obstruction and the expression of 8-OHdG, γ-H2AX, apoptotic proteins and APE was determined. The accumulated p53 activated Bax and caspase-3 7 days after hydronephrosis induction and the resulting high levels of p53-dependent apoptotic proteins and γ-H2AX tended to decrease APE. The intensities of 8-OHdG and caspase-3 immunolocalization significantly increased in obstructed kidneys than in sham-operated kidneys, although APE immunoreactivity increased after hydronephrosis induction. These results suggest that oxidative DNA damages in obstructed kidneys may trigger p53-dependent apoptosis through repression of APE.     

Ciprofloxacin as a potential radio-sensitizer to tumor cells and a radio-protectant for normal cells: differential effects on γ-H2AX formation,p53 phosphorylation,Bcl-2 production,and cell death

Ionizing radiation increases cell mortality in a dose-dependent manner. Increases in DNA double strand breaks, γ-H2AX, p53 phophorylation, and protein levels of p53 and Bax also occur. We investigated the ability of ciprofloxacin (CIP), a widely prescribed antibiotic, to inhibit DNA damage induced by ionizing radiation. Human tumor TK6, NH32 (p53 ^?/? of TK6) cells, and human normal peripheral blood mononuclear cells (PBMCs) were exposed to 2–8 Gy ⁶⁰Co-γ-photon radiation. γ-H2AX (an indicator of DNA strand breaks), phosphorylated p53 (responsible for cell-cycle arrest), Bcl-2 (an apoptotic protein, and cell death were measured. Ionizing irradiation increased γ-H2AX amounts in TK6 cells (p53^+/+) within 1 h in a radiation dose-dependent manner. CIP pretreatment and posttreatment effectively inhibited the increase in γ-H2AX. CIP pretreatment reduced Bcl-2 production but promoted p53 phosphorylation, caspase-3 activation and cell death. In NH32 cells, CIP failed to significantly inhibit the radiation-induced γ-H2AX increase, suggesting that CIP inhibition involves in p53-dependent mechanisms. In normal healthy human PBMCs, CIP failed to block the radiation-induced γ-H2AX increase but effectively increased Bcl-2 production, but blocked the phospho-p53 increase and subsequent cell death. CIP increased Gadd45α, and enhanced p21 protein 24 h postirradiation. Results suggest that CIP exerts its effect in TK6 cells by promoting p53 phosphorylation and inhibiting Bcl-2 production and in PBMCs by inhibiting p53 phosphorylation and increasing Bcl-2 production. Our data are the first to support the view that CIP may be effective to protect normal tissue cells from radiation injury, while enhancing cancer cell death in radiation therapy.     

p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser³⁹². Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.     

Regulation of the human AP-endonuclease (APE1/Ref-1) expression by the tumor suppressor p53 in response to DNA damage

The human AP-endonuclease (APE1/Ref-1), an essential multifunctional protein, plays a central role in the repair of oxidative base damage via the DNA base excision repair (BER) pathway. The mammalian AP-endonuclease (APE1) overexpression is often observed in tumor cells, and confers resistance to various anticancer drugs; its downregulation sensitizes tumor cells to those agents via induction of apoptosis. Here we show that wild type (WT) but not mutant p53 negatively regulates APE1 expression. Time-dependent decrease was observed in APE1 mRNA and protein levels in the human colorectal cancer line HCT116 p53^(+/+), but not in the isogenic p53 null mutant after treatment with camptothecin, a DNA topoisomerase I inhibitor. Furthermore, ectopic expression of WTp53 in the p53 null cells significantly reduced both endogenous APE1 and APE1 promoter-dependent luciferase expression in a dose-dependent fashion. Chromatin immunoprecipitation assays revealed that endogenous p53 is bound to the APE1 promoter region that includes a Sp1 site. We show here that WTp53 interferes with Sp1 binding to the APE1 promoter, which provides a mechanism for the downregulation of APE1. Taken together, our results demonstrate that WTp53 is a negative regulator of APE1 expression, so that repression of APE1 by p53 could provide an additional pathway for p53-dependent induction of apoptosis in response to DNA damage.     

The apoptotic ring: A novel entity with phosphorylated histones H2AX and H2B,and activated DNA damage response kinases

We recently showed that histone H2AX phosphorylated on serine 139 (γ-H2AX), a hallmark of DNA damage response (DDR), also forms early during apoptosis induced by death receptor activation. Here, we extend and discuss our findings on apoptotic γ-H2AX, which differs from the well-established DDR with nuclear foci. During apoptosis induced by death receptors agonists (TRAIL and FasL) and staurosporine, γ-H2AX is initiated in the nuclear periphery immediately inside the nuclear envelope while total H2AX remains distributed throughout the nucleus. This process is readily detectable by immunofluorescence microscopy and we refer to it as the “γ-H2AX ring”. It is conserved both in cancer and normal cells. The γ-H2AX ring contains the activated checkpoints kinases, ATM, Chk2 and DNA-PK; the latter being the main effector for the apoptotic γ-H2AX phosphorylation. Notably, we show here that the γ-H2AX ring coincides with phosphorylated H2B on serine 14 (PS14-H2B), another histone modification associated with apoptosis. The coordinated phosphorylations of H2AX and H2B suggest a previously unrecognized histone phosphorylation signature for apoptosis consisting of γ-H2AX together with PS14-H2B and possibly PY142-H2AX. This signature (“phospho-histone 2 code”) together with the γ-H2AX ring provides a new feature to monitor and study apoptosis.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells

Selective inhibition with sulphonamides of carbonic anhydrase (CA) IX reduces cell proliferation and induces apoptosis in human cancer cells. The effect on CA IX expression of seven previously synthesised sulphonamide inhibitors, with high affinity for CA IX, as well as their effect on the proliferation/apoptosis of cancer/normal cell lines was investigated. Two normal and three human cancer cell lines were used. Treatment resulted in dose- and time-dependent inhibition of the growth of various cancer cell lines. One compound showed remarkably high toxicity towards CA IX-positive HeLa cells. The mechanisms of apoptosis induction were determined with Annexin-V and AO/EB staining, cleaved caspases (caspase-3, caspase-8, caspase-9) and cleaved PARP activation, reactive oxygen species production (ROS), mitochondrial membrane potential (MMP), intracellular pH (pHi), extracellular pH (pHe), lactate level and cell cycle analysis. The autophagy induction mechanisms were also investigated. The modulation of apoptotic and autophagic genes (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin and LC3) was measured using real time PCR. The positive staining using γ-H2AX and AO/EB dye, showed increased cleaved caspase-3, caspase-8, caspase-9, increased ROS production, MMP and enhanced mRNA expression of apoptotic genes, suggesting that anticancer effects are also exerted through its apoptosis-inducing properties. Our results show that such sulphonamides might have the potential as new leads for detailed investigations against CA IX-positive cervical cancers.     

The synergistic effects of valproic acid and fluvastatin on apoptosis induction in glioblastoma multiforme cell lines

Glioblastoma multiforme (GBM) is the most common primary central nervous system malignant tumor. It responds poorly to standard therapies, such as surgical resection, radiation therapy and chemotherapy. Many chemotherapeutic drugs are focused on apoptosis induction and radiation sensitivity. Inhibition of histone acetylation via histone deacetylase inhibitor (HDACI) is one such strategy. Statins (or 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors) are classical drugs used to lower cholesterol but also inhibitors of histone deacetylation activity. This study analyzes the combinatory effects of valproic acid (VPA) and fluvastatin on apoptosis induction in GBM8401 cells. The results show that they act synergistically in inducing γ-H2AX and apoptosis accompanied by higher acetylated histones H3 and H4. Downregulation of p53 occurred by VPA alone and fluvastatin alone, but not at their combined application; upregulation of p21 at the protein level was induced by each of the drugs alone and no further increase occurred at combined application. The drug BEZ235 inhibited phosphorylation of Akt and attenuated the level of γ-H2AX as well as cleaved PARP (cPARP) induced at combined application of VPA and fluvastatin. Induction of apoptosis within a 48 h incubation period was massive when measured as the subG1 peak (97%) and was detected after a 24 h incubation at low level when assayed with PE Annexin V. Synergistic apoptosis induction was demonstrated also after 24 h incubation by the appearance of cPARP. Partial silencing of p21 reduced cPARP as well as the percentage of apoptotic cells in the subG1 peak. However, partial silencing of p53 had no effect on apoptosis. Such findings offer a better understanding of the mechanism of action of HDACIs in combination with statins that may guide the development of a new combinatory reposition for the treatment of GBM.     

Gamma histone 2AX (γ-H2AX)as a predictive tool in radiation oncology

Abstract

Ionizing radiation cause DNA damage to cells, leading them to cell death via DNA double-strand breaks (DSBs) formation. DSBs formation is followed immediately by histone H2AX phosphorylation (γ-H2AX) and multitude repair factors accumulation. Here we present the methods and the bio-sampling for γ-H2AX detection, γ-H2AX formation in normal cells and animal tissues, in cancer cell lines/tissues and in clinical trials after radiation treatment, alone or in combination with other factors. The purpose of this review is to highlight the use of γ-H2AX, as a marker to assess DNA damage and repair.     

Chipping away at γ-H2AX foci

The mammalian histone H2AX protein functions as a dosage-dependent genomic caretaker and tumor suppressor. Phosphorylation of H2AX to form γ-H2AX in chromatin around DNA double strand breaks (DSBs) is an early event following induction of these hazardous lesions. For a decade, mechanisms that regulate H2AX phosphorylation have been investigated mainly through two-dimensional immunofluorescence (IF). We recently used chromatin immunoprecipitation (ChIP) to measure γ-H2AX densities along chromosomal DNA strands broken in G1 phase mouse lymphocytes. Our experiments revealed that (1) γ-H2AX densities in nucleosomes form at high levels near DSBs and at diminishing levels farther and farther away from DNA ends, and (2) ATM regulates H2AX phosphorylation through both MDC1-dependent and MDC1-independent means. Neither of these mechanisms were discovered by previous IF studies due to the inherent limitations of light microscopy. Here, we compare data obtained from parallel γ-H2AX ChIP and three-dimensional IF analyses and discuss the impact of our findings upon molecular mechanisms that regulate H2AX phosphorylation in chromatin around DNA breakage sites.     

Mechanism of UV-Induced Apoptosis in Human Leukemia Cells: Roles of Ca/Mg-Dependent Endonuclease, Caspase-3, and Stress-Activated Protein Kinases

Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. Thein vitroreconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca²⁺/Mg²⁺-dependent, Zn²⁺-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8–7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 μM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 μM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 μM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 μM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca²⁺/Mg²⁺-dependent endonuclease pathway and the caspase-3–PARP cleavage–Ca²⁺/Mg²⁺-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.     

Activation of DNA damage response pathways in human mesenchymal stem cells exposed to cisplatin or γ-irradiation

DNA damaging agents are widely used in treatment of hematogical malignancies and solid tumors. While effects on hematopoietic stem cells have been characterized, less is known about the DNA damage response in human mesenchymal stem cells (hMSCs) in the bone marrow stroma, progenitors of osteoblasts, chondrocytes and adipocytes. To elucidate the response of undifferentiated hMSCs to γ-irradiation and cisplatin, key DNA damage responses have been characterised in hMSCs from normal adult donors. Cisplatin and γ-irradiation activated the DNA damage response in hMSCs, including induction of p53 and p21, and activation of PI3 kinase-related protein kinase (PIKK)-dependent phosphorylation of histone H2AX on serine 139, and replication protein A2 on serine4/serine8. Chemical inhibition of ATM or DNA-PK reduced DNA damage-induced phosphorylation of H2AX, indicating a role for both PIKKs in the response of hMSCs to DNA damage. Consistent with repair of DNA strand breaks, γ-H2AX staining decreased by 24 hours following gamma-irradiation. γ-irradiation arrested hMSCs in the G₁ phase of the cell cycle, while cisplatin induced S-phase arrest, mediated in part by the ATR/Chk1 checkpoint pathway. In hMSCs isolated from a chronic lymphocytic leukemia (CLL) patient, p53 and p21 were induced by cisplatin and γ-irradiation, while RPA2 was phosphorylated on serine4/8 in particular following cisplatin. Compared to peripheral blood lymphocytes or the leukemia cell line K562, both normal hMSCs and CLL-derived hMSCs were more resistant to cisplatin and γ-irradiation. These results provide insights into key pathways mediating the response of bone marrow-derived hMSCs to DNA damaging agents used in cancer treatment.     

An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis

The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.     

COX-2 regulates the proliferation of glioma stem like cells

Cancer stem-like cells (CSCs) possessing features of neural precursor cells (NPC) influence initiation, recurrence and chemoresistance of glioblastoma multiforme (GBM). As inflammation is crucial for glioblastoma progression we investigated the effect of chronic IL-1β treatment on CSCs derived from glioblastoma cell line U87MG. Exposure to IL-1β for 10 days increased (i) accumulation of 8-OHdG - a key biomarker of oxidative DNA damage; (ii) DNA damage response (DDR) indicators γH2AX, ATM and DNA-PK; (iii) nuclear and cytoplasmic p53 and COX-2 levels and (iv) interaction between COX-2 and p53. Despite upregulating p53 expression IL-1β had no effect on cell cycle progression, apoptosis or self renewal capacity of CSCs. COX-2 inhibitor Celecoxib reduced self renewal capacity and increased apoptosis of both control and IL-1β treated CSCs. Therefore the ability of COX-2 to regulate proliferation of CSCs irrespective of exposure to IL-1β, warrants further investigation of COX-2 as a potential anti-glioma target.     

Caspase-2 activation in neural stem cells undergoing oxidative stress-induced apoptosis

Oxidative stress occurs as a consequence of disturbance in the balance between the generation of reactive oxygen species (ROS) and the antioxidant defence mechanisms. The interaction of ROS with DNA can cause single-, or double-strand breaks that subsequently can lead to the activation of p53, which is central for the regulation of cellular response, e.g. apoptosis, to a range of environmental and intracellular stresses. Previous reports have suggested a regulatory role of p53 in the early activation of caspase-2, upstream of mitochondrial apoptotic signaling. Here we show that excessive ROS formation, induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) exposure, induces apoptosis in primary cultured neural stem cells (NSCs) from cortices of E15 rat embryos. Following DMNQ exposure cells exhibited apoptotic hallmarks such as Bax oligomerization and activation, cytochrome c release, caspase activation and chromatin condensation. Additionally, we could show early p53 accumulation and a subsequent activation of caspase-2. The attenuation of caspase-2 activity with selective inhibitors could antagonize the mitochondrial signaling pathway and cell death. Overall, our results strongly suggest that DMNQ-induced oxidative stress causes p53 accumulation and consequently caspase-2 activation, which in turn initiates apoptotic cell death via the mitochondria-mediated caspase-dependent pathway in NSCs.     

hMTH1 depletion promotes oxidative-stress-induced apoptosis through a Noxa- and caspase-3/7-mediated signaling pathway

Although the accumulation of 8-oxo-dGTP in DNA is associated with apoptotic cell death and mutagenesis, little is known about the exact mechanism of hMTH1-mediated suppression of oxidative-stress-induced cell death. Therefore, we investigated the regulation of DNA-damage-related apoptosis induced by oxidative stress using control and hMTH1 knockdown cells. Small interfering RNA (siRNA) was used to suppress hMTH1 expression in p53-proficient GM00637 and H460 cells, resulting in a significant increase in apoptotic cell death after H(2)O(2) exposure; however, p53-null, hMTH1-deficient H1299 cells did not exhibit H(2)O(2)-induced apoptosis. In addition, hMTH1-deficient GM00637 and H460 cells showed increased caspase-3/7 activity, cleaved caspase-8, and Noxa expression, and gamma-H2AX formation in response to H(2)O(2). In contrast, the caspase inhibitors, p53-siRNA, and Noxa-siRNA suppressed H(2)O(2)-induced cell death. Moreover, in 8-week (long-term) cultured H460 and H1299 cells, hMTH1 suppression increased cell death, Noxa expression, and gamma-H2AX after H(2)O(2) exposure, compared to 3-week (short-term) cultured cells. These data indicate that hMTH1 plays an important role in protecting cells against H(2)O(2)-induced apoptosis via a Noxa- and caspase-3/7-mediated signaling pathway, thus conferring a survival advantage through the inhibition of oxidative-stress-induced DNA damage.