Effect of Functional Inhibition of BACE1 on Sensitization to γ-Irradiation in Cancer Cells.

Developing strategies for the radiosensitization of cancer cells by the inhibition of genes, which harbor low toxicity to normal cells, will be useful for improving cancer radiotherapy. Here, we focused on a ß-site of amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1; ß-secretase, memapsin-2). By functional inhibition of this peptidase by siRNA, it has also recently been shown that the DNA strand break marker, γH2AX foci, increased, suggesting its involvement in DNA damage response. To investigate this possibility, we knocked down BACE1 with siRNA in cancer cell lines, and sensitization to γ-irradiation was examined by a colony formation assay, γH2AX foci and level analysis, and flow cytometry. BACE1 knockdown resulted in the sensitization of HeLa, MDA-MB-231, U2OS, and SAOS cells to γ-irradiation in a diverse range. BACE1 knockdown showed a weak radiosensitization effect in osteosarcoma U2OS cells, which has a normal p53 function. HeLa and SAOS cells, which harbor p53 dysfunction, exhibited a greater level of radiosensitization. These results suggest that BACE1 may be a potential target for the radiosensitization in particular cancer cells.

Radiosensitization to γ-Ray by Functional Inhibition of APOBEC3G.

The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue. In this study, we performed a comprehensive screening of radiosensitization targets in human lung cancer cell line A549 using an shRNA library and identified apolipoprotein B mRNA editing enzyme catalytic subunit 3G (APOBEC3G: A3G) as a candidate target. APOBEC3G is an innate restriction factor that inhibits HIV-1 infection as a cytidine deaminase. APOBEC3G knockdown with siRNA showed an increased radiosensitivity in several cancer cell lines, including pancreatic cancer MIAPaCa2 cells and lung cancer A549 cells. Cell cycle analysis revealed that APOBEC3G knockdown increased S-phase arrest in MIAPaCa2 and G2/M arrest in A549 cells after γ-irradiation. DNA double-strand break marker γH2AX level was increased in APOBEC3G-knocked-down MIAPaCa2 cells after γ-irradiation. Using a xenograft model of A549 in mice, enhanced radiosensitivity by a combination of X-ray irradiation and APOBEC3G knockdown was observed. These results suggest that the functional inhibition of APOBEC3G sensitizes cancer cells to radiation by attenuating the activation of the DNA repair pathway, suggesting that APOBEC3G could be useful as a target for the radiosensitization of cancer therapy.

A newly established monoclonal antibody against ERCC1 detects major isoforms of ERCC1 in gastric cancer.

Identifying patients resistant to cisplatin treatment is expected to improve cisplatin-based chemotherapy for various types of cancers. Excision repair cross-complementing group 1 (ERCC1) is involved in several repair processes of cisplatin-induced DNA crosslinks. ERCC1 overexpression is reported as a candidate prognostic factor and considered to cause cisplatin resistance in major solid cancers. However, anti-ERCC1 antibodies capable of evaluating expression levels of ERCC1 in clinical specimens were not fully optimized. A mouse monoclonal antibody against human ERCC1 was generated in this study. The developed antibody 9D11 specifically detected isoforms of 201, 202, 203 but not 204, which lacks the exon 3 coding region. To evaluate the diagnostic usefulness of this antibody, we have focused on gastric cancer because it is one of the major cancers in Japan. When ERCC1 expression was analyzed in seventeen kinds of human gastric cancer cell lines, all the cell lines were found to express either 201, 202, and/or 203 as major isoforms of ERCC1, but not 204 by Western blotting analysis. Immunohistochemical staining showed that ERCC1 protein was exclusively detected in nuclei of the cells and a moderate level of constant positivity was observed in nuclei of vascular endothelial cells. It showed a clear staining pattern in clinical specimens of gastric cancers. Antibody 9D11 may thus be useful for estimating expression levels of ERCC1 in clinical specimens.

Dysfunction of Poly (ADP-Ribose) Glycohydrolase Induces a Synthetic Lethal Effect in Dual Specificity Phosphatase 22-Deficient Lung Cancer Cells.

Poly (ADP-ribose) glycohydrolase (PARG) is the main enzyme responsible for catabolism of poly (ADP-ribose) (PAR), synthesized by PARP. PARG dysfunction sensitizes certain cancer cells to alkylating agents and cisplatin by perturbing the DNA damage response. The gene mutations that sensitize cancer cells to PARG dysfunction-induced death remain to be identified. Here, we performed a comprehensive analysis of synthetic lethal genes using inducible PARG knockdown cells and identified dual specificity phosphatase 22 (DUSP22) as a novel synthetic lethal gene related to PARG dysfunction. DUSP22 is considered a tumor suppressor and its mutation has been frequently reported in lung, colon, and other tumors. In the absence of DNA damage, dual depletion of PARG and DUSP22 in HeLa and lung cancer A549 cells reduced survival compared with single-knockdown counterparts. Dual depletion of PARG and DUSP22 increased the apoptotic sub-G1 fraction and upregulated PUMA in lung cancer A549, PC14, and SBC5 cells, and inhibited the PI3K/AKT/mTOR pathway in A549 cells, suggesting that dual depletion of PARG and DUSP22 induced apoptosis by upregulating PUMA and suppressing the PI3K/AKT/mTOR pathway. Consistently, the growth of tumors derived from double knockdown A549 cells was slower compared with those derived from control siRNA-transfected cells. Taken together, these results indicate that DUSP22 deficiency exerts a synthetic lethal effect when combined with PARG dysfunction, suggesting that DUSP22 dysfunction could be a useful biomarker for cancer therapy using PARG inhibitors. SIGNIFICANCE: This study identified DUSP22 as a novel synthetic lethal gene under the condition of PARG dysfunction and elucidated the mechanism of synthetic lethality in lung cancer cells.

Concomitant administration of radiation with eribulin improves the survival of mice harboring intracerebral glioblastoma.

Glioblastoma is the most common and devastating type of malignant brain tumor. We recently found that eribulin suppresses glioma growth in vitro and in vivo and that eribulin is efficiently transferred into mouse brain tumors at a high concentration. Eribulin is a non-taxane microtubule inhibitor approved for breast cancer and liposarcoma. Cells arrested in M-phase by chemotherapeutic agents such as microtubule inhibitors are highly sensitive to radiation-induced DNA damage. Several recent case reports have demonstrated the clinical benefits of eribulin combined with radiation therapy for metastatic brain tumors. In this study, we investigated the efficacy of a combined eribulin and radiation treatment on human glioblastoma cells. The glioblastoma cell lines U87MG, U251MG and U118MG, and SJ28 cells, a patient-derived sphere culture cell line, were used to determine the radiosensitizing effect of eribulin using western blotting, flow cytometry and clonogenic assay. Subcutaneous and intracerebral glioma xenografts were generated in mice to assess the efficacy of the combined treatment. The combination of eribulin and radiation enhanced DNA damage in vitro. The clonogenic assay of U87MG demonstrated the radiosensitizing effect of eribulin. The concomitant eribulin and radiation treatment significantly prolonged the survival of mice harboring intracerebral glioma xenografts compared with eribulin or radiation alone (P < .0001). In addition, maintenance administration of eribulin after the concomitant treatment further controlled brain tumor growth. Aberrant microvasculature was decreased in these tumors. Concomitant treatment with eribulin and radiation followed by maintenance administration of eribulin may serve as a novel therapeutic strategy for glioblastomas.

PARG Inhibitors and Functional PARG Inhibition Models.

Poly(ADP-ribose) polymerases (PARPs) family proteins catalyze poly(ADP-ribosylation) (PARylation) by conjugating ADP-ribose residues repeatedly on amino acid residues using nicotinamide adenine dinucleotide as a substrate. The inhibitors of PARP widely block DNA repair processes and are currently examined in clinical trials of cancer therapy. Poly(ADP-ribose) glycohydrolase (PARG) is the main nuclear enzyme, which digests poly(ADP-ribose) into ADP-ribose. PARG inhibitor could also be considered as a chemotherapeutic agent for cancer, because of its involvement in DNA repair. Various PARG inhibitors with IC50 value of micromolar to submicromolar range have been reported. However, for most of these chemicals, the specificity of inhibition has not been fully evaluated. PARG functional inhibition models in various organisms have been developed. Here, inducible PARG knockdown system was developed in HeLa cells and the cell line will be useful for identifying the synthetic lethal genes or affecting genes for PARG inhibitor treatment and also for functional elucidation of PARP superfamily molecules.

Radiosensitization by PARP inhibition to proton beam irradiation in cancer cells.

The poly(ADP-ribose) polymerase (PARP)-1 regulates DNA damage responses and promotes base excision repair. PARP inhibitors have been shown to enhance the cytotoxicity of ionizing radiation in various cancer cells and animal models. We have demonstrated that the PARP inhibitor (PARPi) AZD2281 is also an effective radiosensitizer for carbon-ion radiation; thus, we speculated that the PARPi could be applied to a wide therapeutic range of linear energy transfer (LET) radiation as a radiosensitizer. Institutes for biological experiments using proton beam are limited worldwide. This study was performed as a cooperative research at heavy ion medical accelerator in Chiba (HIMAC) in National Institute of Radiological Sciences. HIMAC can generate various ion beams; this enabled us to compare the radiosensitization effect of the PARPi on cells subjected to proton and carbon-ion beams from the same beam line. After physical optimization of proton beam irradiation, the radiosensitization effect of the PARPi was assessed in the human lung cancer cell line, A549, and the pancreatic cancer cell line, MIA PaCa-2. The effect of the PARPi, AZD2281, on radiosensitization to Bragg peak was more significant than that to entrance region. The PARPi increased the number of phosphorylated H2AX (γ-H2AX) foci and enhanced G2/M arrest after proton beam irradiation. This result supports our hypothesis that a PARPi could be applied to a wide therapeutic range of LET radiation by blocking the DNA repair response.

Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo.

Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

Loss of CtIP disturbs homologous recombination repair and sensitizes breast cancer cells to PARP inhibitors.

Breast cancer is one of the leading causes of death worldwide, and therefore, new and improved approaches for the treatment of breast cancer are desperately needed. CtIP (RBBP8) is a multifunctional protein that is involved in various cellular functions, including transcription, DNA replication, DNA repair and the G1 and G2 cell cycle checkpoints. CtIP plays an important role in homologous recombination repair by interacting with tumor suppressor protein BRCA1. Here, we analyzed the expression profile of CtIP by data mining using published microarray data sets. We found that CtIP expression is frequently decreased in breast cancer patients, and the patient group with low-expressing CtIP mRNA is associated with a significantly lower survival rate. The knockdown of CtIP in breast cancer MCF7 cells reduced Rad51 foci numbers and enhanced f H2AX foci formation after f-irradiation, suggesting that deficiency of CtIP decreases homologous recombination repair and delays DNA double strand break repair. To explore the effect of CtIP on PARP inhibitor therapy for breast cancer, CtIP-depleted MCF7 cells were treated with PARP inhibitor olaparib (AZD2281) or veliparib (ABT-888). As in BRCA mutated cells, PARP inhibitors showed cytotoxicity to CtIP-depleted cells by preventing cells from repairing DNA damage, leading to decreased cell viability. Further, a xenograft tumor model in mice with MCF7 cells demonstrated significantly increased sensitivity towards PARP inhibition under CtIP deficiency. In summary, this study shows that low level of CtIP expression is associated with poor prognosis in breast cancer, and provides a rationale for establishing CtIP expression as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients.

A comprehensive analysis of radiosensitization targets; functional inhibition of DNA methyltransferase 3B radiosensitizes by disrupting DNA damage regulation.

A comprehensive genome-wide screen of radiosensitization targets in HeLa cells was performed using a shRNA-library/functional cluster analysis and DNMT3B was identified as a candidate target. DNMT3B RNAi increased the sensitivity of HeLa, A549 and HCT116 cells to both γ-irradiation and carbon-ion beam irradiation. DNMT3B RNAi reduced the activation of DNA damage responses induced by γ-irradiation, including HP1ß-, γH2AX- and Rad51-foci formation. DNMT3B RNAi impaired damage-dependent H2AX accumulation and showed a reduced level of γH2AX induction after γ-irradiation. DNMT3B interacted with HP1ß in non-irradiated conditions, whereas irradiation abrogated the DNMT3B/HP1ß complex but induced interaction between DNMT3B and H2AX. Consistent with radiosensitization, TP63, BAX, PUMA and NOXA expression was induced after γ-irradiation in DNMT3B knockdown cells. Together with the observation that H2AX overexpression canceled radiosensitization by DNMT3B RNAi, these results suggest that DNMT3B RNAi induced radiosensitization through impairment of damage-dependent HP1ß foci formation and efficient γH2AX-induction mechanisms including H2AX accumulation. Enhanced radiosensitivity by DNMT3B RNAi was also observed in a tumor xenograft model. Taken together, the current study implies that comprehensive screening accompanied by a cluster analysis enabled the identification of radiosensitization targets. Downregulation of DNMT3B, one of the targets identified using this method, radiosensitizes cancer cells by disturbing multiple DNA damage responses.

Proteomic analysis of cellular response induced by boron neutron capture reaction in human squamous cell carcinoma SAS cells.

To understand the mechanism of cell death induced by boron neutron capture reaction (BNCR), we performed proteome analyses of human squamous tumor SAS cells after BNCR. Cells were irradiated with thermal neutron beam at KUR after incubation under boronophenylalanine (BPA)(+) and BPA(-) conditions. BNCR mainly induced typical apoptosis in SAS cells 24h post-irradiation. Proteomic analysis in SAS cells suggested that proteins functioning in endoplasmic reticulum, DNA repair, and RNA processing showed dynamic changes at early phase after BNCR and could be involved in the regulation of cellular response to BNCR. We found that the BNCR induces fragments of endoplasmic reticulum-localized lymphoid-restricted protein (LRMP). The fragmentation of LRMP was also observed in the rat tumor graft model 20 hours after BNCT treatment carried out at the National Nuclear Center of the Republic of Kazakhstan. These data suggest that dynamic changes of LRMP could be involved during cellular response to BNCR.

Parp-1 deficiency in ES cells promotes invasive and metastatic lesions accompanying induction of trophoblast giant cells during tumorigenesis in uterine environment.

Embryonic stem (ES) cells deficient in poly(ADP-ribose) polymerase-1 (Parp-1) develop into teratocarcinomas with the appearance of trophoblast giant cells (TGCs) when injected subcutaneously into nude mice. Because the uterus is one of the original organs in which germ cell tumors develop with induction of trophoblast lineage, here we investigated whether Parp-1 deficiency in ES cells affects teratocarcinoma formation processes by grafting ES cells into the horns of uteri. Teratocarcinomas developed from both wild-type (Parp-1(+/+) ) and Parp-1(-/-) ES cells. The weights of the tumors derived from Parp-1(-/-) ES cells were lower than those of the tumors derived from Parp-1(+/+) ES cells (P < 0.05). The Parp-1(-/-) tumors showed the appearance of TGCs. Notably, organ metastasis to the lung and liver was observed for the Parp-1(-/-) tumors, but not for the Parp-1(+/+) tumors (P < 0.05). Invasions were more frequently observed with the Parp-1(-/-) tumors compared with the Parp-1(+/+) tumors (P < 0.05). Since TGCs are known to have invasive properties, the appearance of TGCs may have supported the metastatic process. The present findings suggest that loss of Parp-1 during teratocarcinoma formation might augment invasive and metastatic properties of the tumors in the uterine environment.

The H19 induction triggers trophoblast lineage commitment in mouse ES cells.

Trophoblast lineage differentiation is properly regulated to support embryogenesis. Besides normal developmental process, during germ cell tumor formation or development of other reproductive system diseases, unregulated trophoblast differentiation is also observed and affects the pathogenesis of the diseases. During normal embryogenesis, cell fate of late-stage blastcyst is regulated by a reciprocal repression of the key transcriptional factors; Oct3/4 dominancy inhibits Cdx2 expression in inner cell mass (ICM) and leads them to epiblast/primitive ectoderm but Cdx2 dominancy in trophectoderm (TE) leads them to trophoblast lineage. In contrast during early blastcyst stage, the Cdx2 expression is restricted in TE and not present in ICM, although Oct3/4 signaling does not inhibit the Cdx2 expression in ICM, implying that some factors could be inactivated leading to the suppressed Cdx2 expression in ICM of early blastcyst. ES cells (ESCs), which are derived from ICM, could be a unique model to study trophoblast differentiation in an ectopic context. We previously showed that poly(ADP-ribose) polymerase-1 (Parp-1) deficient ESCs highly expressed non-coding RNA H19 and could differentiate into trophoblast lineage. The expression of H19 is known to start at pre-blastcyst stage during mouse development, and the gene shows high expression only in trophoectoderm (TE) at blastcyst stage. However, its role in trophoblast differentiation has not been clarified yet. Thus, we hypothesized that the H19 activation may act as a trigger for induction of trophoblast differentiation cascade in mouse ESCs. To investigate this issue, we asked whether a forced H19 expression drives ESCs into trophoblast lineage or not. We demonstrated that the H19 induction leads to trophoblast lineage commitment through induction of the Cdx2 expression. We also showed that the expression of Cdx2 is induced in ESCs by forced H19 expression even under a high level of Oct3/4, which could act as a suppressor for Cdx2 expression. It is thus suggested that the H19 induction promotes trophoblast lineage commitment against the repression pressure by Oct3/4 in differentiating ESCs. Taken together, this study suggests that the H19 expression is able to function as a cascade activator of trophoblast lineage commitment possibly by overriding the Oct3/4 action in ESCs.

Poly(ADP-ribosyl)ation in carcinogenesis.

Cancer develops through diverse genetic, epigenetic and other changes, so-called 'multi-step carcinogenesis', and each cancer harbors different alterations and properties. Here in this article we review how poly(ADP-ribosyl)ation is involved in multi-step and diverse pathways of carcinogenesis. Involvement of poly- and mono-ADP-ribosylation in carcinogenesis has been studied at molecular and cellular levels, and further by animal models and human genetic approaches. PolyADP-ribosylation acts in DNA damage repair response and maintenance mechanisms of genomic stability. Several DNA repair pathways, including base-excision repair and double strand break repair pathways, involve PARP and PARG functions. These care-taker functions of poly(ADP-ribosyl)ation suggest that polyADP-ribosyation may mainly act in a tumor suppressive manner because genomic instability caused by defective DNA repair response could serve as a driving force for tumor progression, leading to invasion, metastasis and relapse of cancer. On the other hand, the new concept of 'synthetic lethality by PARP inhibition' suggests the significance of PARP activities for survival of cancer cells that harbor defects in DNA repair. Accumulating evidence has revealed that some PARP family molecules are involved in various signaling cascades other than DNA repair, including epigenetic and transcriptional regulations, inflammation/immune response and epithelial-mesenchymal transition, suggesting that poly(ADP-ribosyl)ation both promotes and suppresses carcinogenic processes depending on the conditions. Expanding understanding of poly(ADP-ribosyl)ation suggests that strategies to achieve cancer prevention targeting poly(ADP-ribosyl)ation for genome protection against life-long exposure to environmental carcinogens and endogenous carcinogenic stimuli.

Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation.

Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/µm) and Fe-ion irradiation (200 keV/µm) were also examined. Parg(-/-) cells were more sensitive to LET 70 keV/µm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on DNA double strand break repair responses and enhanced cell death.

The Arf/p53 protein module, which induces apoptosis, down-regulates histone H2AX to allow normal cells to survive in the presence of anti-cancer drugs.

BACKGROUND: It is unclear how DNA-damaging agents target cancer cells over normal somatic cells. RESULTS: Arf/p53-dependent down-regulation of H2AX enables normal cells to survive after DNA damage. CONCLUSION: Transformed cells, which harbor mutations in either Arf or p53, are more sensitive to DNA-damaging agents. SIGNIFICANCE: Cellular transformation renders cells more susceptible to some DNA-damaging agents. Anti-cancer drugs generally target cancer cells rather than normal somatic cells. However, the factors that determine this differential sensitivity are poorly understood. Here we show that Arf/p53-dependent down-regulation of H2AX induced the selective survival of normal cells after drug treatment, resulting in the preferential targeting of cancer cells. Treatment with camptothecin, a topoisomerase I inhibitor, caused normal cells to down-regulate H2AX and become quiescent, a process mediated by both Arf and p53. In contrast, transformed cells that harbor mutations in either Arf or p53 do not down-regulate H2AX and are more sensitive to drugs unless they have developed drug resistance. Such transformation-associated changes in H2AX expression rendered cancer cells more susceptible to drug-induced damage (by two orders of magnitude). Thus, the expression of H2AX and γH2AX (phosphorylated form of H2AX at Ser-139) is a critical factor that determines drug sensitivity and should be considered when administering chemotherapy.

Rapid degradation of poly(ADP-ribose) after injection into the mouse bloodstream.

Extensive DNA damage leads to the activation of poly(ADP-ribose) polymerase and subsequently to the formation of poly(ADP-ribose). When the damage is severe or leads to cell death, poly(ADP-ribose) may leak into the blood circulation. The metabolism of poly(ADP-ribose) in the bloodstream is not well understood. Thus, in the present study, the metabolism of P-labeled poly(ADP-ribose) was followed in mice after injection of this labeled compound into the tail vein. The results showed that 5 min after injection more than half of the radioactivity was concentrated in acid-soluble fractions, namely in low molecular weight compounds in the blood, liver, and kidneys. Most of this radioactivity was in the form of inorganic phosphate, detected 5 min post-injection in the blood, kidneys, and urine. By contrast, the metabolites ADP-ribose and phosphoribosyl-AMP were not detected in any of the tissues nor in blood or urine. Taken together, these findings suggest that once poly(ADP-ribose) enters the bloodstream it is rapidly degraded, thereby preventing its accumulation in the blood.

Induction of cancerous stem cells during embryonic stem cell differentiation.

Stem cell maintenance depends on their surrounding microenvironment, and aberrancies in the environment have been associated with tumorigenesis. However, it remains to be elucidated whether an environmental aberrancy can act as a carcinogenic stress for cellular transformation of differentiating stem cells into cancer stem cells. Here, utilizing mouse embryonic stem cells as a model, it was illustrated that environmental aberrancy during differentiation leads to the emergence of pluripotent cells showing cancerous characteristics. Analogous to precancerous stages, DNA lesions were spontaneously accumulated during embryonic stem cell differentiation under aberrational environments, which activates barrier responses such as senescence and apoptosis. However, overwhelming such barrier responses, piled-up spheres were subsequently induced from the previously senescent cells. The sphere cells exhibit aneuploidy and dysfunction of the Arf-p53 module as well as enhanced tumorigenicity and a strong self-renewal capacity, suggesting development of cancerous stem cells. Our current study suggests that stem cells differentiating in an aberrational environment are at risk of cellular transformation into malignant counterparts.

Radiosensitization effect of poly(ADP-ribose) polymerase inhibition in cells exposed to low and high liner energy transfer radiation.

Poly(ADP-ribose) polymerase (PARP)-1 promotes base excision repair and DNA strand break repair. Inhibitors of PARP enhance the cytotoxic effects of γ-irradiation and X-irradiation. We investigated the impact of PARP inhibition on the responses to γ-irradiation (low liner energy transfer [LET] radiation) and carbon-ion irradiation (high LET radiation) in the human pancreatic cancer cell line MIA PaCa-2. Cell survival was assessed by colony formation assay after combination treatment with the PARP inhibitor AZD2281 and single fraction γ-irradiation and carbon-ion irradiation (13 and 70 keV/µm [LET 13 and LET 70]). The DNA damage response (DDR) was assessed by pulse field gel electrophoresis, western blotting and flow cytometry. Treatment with a PARP inhibitor enhanced the cytotoxic effect of γ-irradiation and LET 13 and LET 70 carbon-ion irradiation. Moreover, the radiosensitization effect was greater for LET 70 than for LET 13 irradiation. Prolonged and increased levels of γ-H2AX were observed both after γ-irradiation and carbon-ion irradiation in the presence of the PARP inhibitor. Enhanced level of phosphorylated-p53 (Ser-15) was observed after γ-irradiation but not after carbon-ion irradiation. PARP inhibitor treatment induced S phase arrest and enhanced subsequent G2/M arrest both after γ-irradiation and carbon-ion irradiation. These results suggest that the induction of S phase arrest through an enhanced DDR and a local delay in DNA double strand break processing by PARP inhibition caused sensitization to γ-irradiation and carbon-ion irradiation. Taken together, PARP inhibitors might be applicable to a wide therapeutic range of LET radiation through their effects on the DDR.

Onset of quiescence following p53 mediated down-regulation of H2AX in normal cells.

Normal cells, both in vivo and in vitro, become quiescent after serial cell proliferation. During this process, cells can develop immortality with genomic instability, although the mechanisms by which this is regulated are unclear. Here, we show that a growth-arrested cellular status is produced by the down-regulation of histone H2AX in normal cells. Normal mouse embryonic fibroblast cells preserve an H2AX diminished quiescent status through p53 regulation and stable-diploidy maintenance. However, such quiescence is abrogated under continuous growth stimulation, inducing DNA replication stress. Because DNA replication stress-associated lesions are cryptogenic and capable of mediating chromosome-bridge formation and cytokinesis failure, this results in tetraploidization. Arf/p53 module-mutation is induced during tetraploidization with the resulting H2AX recovery and immortality acquisition. Thus, although cellular homeostasis is preserved under quiescence with stable diploidy, tetraploidization induced under growth stimulation disrupts the homeostasis and triggers immortality acquisition.