Physiological levels of poly(ADP-ribose) during the cell cycle regulate HeLa cell proliferation.

Protein targets of polyADP-ribosylation undergo covalent modification with high-molecular-weight, branched poly(ADP-ribose) (PAR) of lengths up to 200 or more ADP-ribose residues derived from NAD+. PAR polymerase 1 (PARP1) is the most abundant and well-characterized enzyme involved in PAR biosynthesis. Extensive studies have been carried out to determine how polyADP-ribosylation (PARylation) regulates cell proliferation during cell cycle, with conflicting conclusions. Since significant activation of PARP1 occurs during cell lysis in vitro, we changed the standard method for cell lysis, and using our sensitive ELISA system, quantified without addition of a PAR glycohydrolase inhibitor and clarified that the PAR level is significantly higher in S phase than that in G1. Under normal condition in the absence of exogenous DNA-damaging agent, PAR turns over with a half-life of <40 s; consistent with significant decrease of NAD+ levels in S phase, which is rescued by PARP inhibitors, in line with the observed rapid turnover of PAR. PARP inhibitors delayed cell cycle in S phase and decreased cell proliferation. Our results underscore the importance of a suitable assay system to measure rapid PAR chain dynamics in living cells and aid our understanding of the function of PARylation during the cell cycle.

Elucidating Differences in Early-Stage Centrosome Amplification in Primary and Immortalized Mouse Cells.

The centrosome is involved in cytoplasmic microtubule organization during interphase and in mitotic spindle assembly during cell division. Centrosome amplification (abnormal proliferation of centrosome number) has been observed in several types of cancer and in precancerous conditions. Therefore, it is important to elucidate the mechanism of centrosome amplification in order to understand the early stage of carcinogenesis. Primary cells could be used to better understand the early stage of carcinogenesis rather than immortalized cells, which tend to have various genetic and epigenetic changes. Previously, we demonstrated that a poly(ADP-ribose) polymerase (PARP) inhibitor, 3-aminobenzamide (3AB), which is known to be nontoxic and nonmutagenic, could induce centrosome amplification and chromosomal aneuploidy in CHO-K1 cells. In this study, we compared primary mouse embryonic fibroblasts (MEF) and immortalized MEF using 3AB. Although centrosome amplification was induced with 3AB treatment in immortalized MEF, a more potent PARP inhibitor, AG14361, was required for primary MEF. However, after centrosome amplification, neither 3AB in immortalized MEF nor AG14361 in primary MEF caused chromosomal aneuploidy, suggesting that further genetic and/or epigenetic change(s) are required to exhibit aneuploidy. The DNA-damaging agents doxorubicin and γ-irradiation can cause cancer and centrosome amplification in experimental animals. Although doxorubicin and γ-irradiation induced centrosome amplification and led to decreased p27Kip protein levels in immortalized MEF and primary MEF, the phosphorylation ratio of nucleophosmin (Thr199) increased in immortalized MEF, whereas it decreased in primary MEF. These results suggest that there exists a yet unidentified pathway, different from the nucleophosmin phosphorylation pathway, which can cause centrosome amplification in primary MEF.

PARP Inhibitor Decreases Akt Phosphorylation and Induces Centrosome Amplification and Chromosomal Aneuploidy in CHO-K1 Cells.

Cancer cells are known to have chromosomal number abnormalities (aneuploidy), a hallmark of malignant tumors. Cancer cells also have an increased number of centrosomes (centrosome amplification). Paradoxically, cancer therapies, including γ-irradiation and some anticancer drugs, are carcinogenic and can induce centrosome amplification and chromosomal aneuploidy. Thus, the processes of carcinogenesis and killing cancer cells might have some mechanisms in common. Previously, we found that the inhibitors of polyADP-ribosylation, a post-translational modification of proteins, caused centrosome amplification. However, the mechanism of action of the inhibitors of polyADP-ribosylation is not fully understood. In this study, we found that an inhibitor of polyADP-ribosylation, 3-aminobenzamide, caused centrosome amplification, as well as aneuploidy of chromosomes in CHO-K1 cells. Moreover, inhibitors of polyADP-ribosylation inhibited AKT phosphorylation, and inhibitors of AKT phosphorylation inhibited polyADP-ribosylation, suggesting the involvement of polyADP-ribosylation in the PI3K/Akt/mTOR signaling pathway for controlling cell proliferation. Our data suggest a possibility for developing drugs that induce centrosome amplification and aneuploidy for therapeutic applications to clinical cancer.

ADP-Ribosylation as Post-Translational Modification of Proteins: Use of Inhibitors in Cancer Control.

Among the post-translational modifications of proteins, ADP-ribosylation has been studied for over fifty years, and a large set of functions, including DNA repair, transcription, and cell signaling, have been assigned to this post-translational modification (PTM). This review presents an update on the function of a large set of enzyme writers, the readers that are recruited by the modified targets, and the erasers that reverse the modification to the original amino acid residue, removing the covalent bonds formed. In particular, the review provides details on the involvement of the enzymes performing monoADP-ribosylation/polyADP-ribosylation (MAR/PAR) cycling in cancers. Of note, there is potential for the application of the inhibitors developed for cancer also in the therapy of non-oncological diseases such as the protection against oxidative stress, the suppression of inflammatory responses, and the treatment of neurodegenerative diseases. This field of studies is not concluded, since novel enzymes are being discovered at a rapid pace.

Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells.

Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24-72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX, PAR could be a sensitive marker for DNA single- and double-strand breaks. These two molecular markers provide evidence of physiological changes occurring within cells.

An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.

Lectin Microarray-Based Sero-Biomarker Verification Targeting Aberrant O-Linked Glycosylation on Mucin 1.

Glycoform of mucin 1 (MUC1) in cancerous cells changes markedly with cell differentiation, and thus, qualitative detection and verification of the MUC1 glycosylation changes have potential diagnostic value. We have developed an ultrasensitive method to detect the changes in cholangiocarcinoma (CC), which produces MUC1, and applied it in the diagnostics development. The focused glycan analysis using 43-lectin-immobilized microarray could obtain the glycan profiles of sialylated MUC1 in 5 µL of sera. The high-throughput analysis detected disease-specific alterations of glycosylation, and the statistical analysis confirmed that use of Wisteria floribunda agglutinin (WFA) alone produced a diagnostic score sufficient for discriminating 33 CC cases from 40 hepatolithiasis patients and 48 normal controls (p < 0.0001). The CC-related glycosylation change was verified by the lectin-antibody sandwich ELISA with WFA in two cohorts: (1) 78 Opisthorchis viverrini infected patients without CC and 78 with CC, (2) 33 CC patients and 40 hepatolithiasis patients (the same cohort used for the above lectin microarray). The WFA positivity distinguished patients with CC (opisthorchiasis: p < 0.0001, odds ratio = 1.047; hepatolithiasis: p = 0.0002, odds ratio = 1.018). Sensitive detection of qualitative alterations of sialylated MUC1 glycosylation is indispensable for the development of our glycodiagnostic test for CC.

Inhibition of Crm1-p53 interaction and nuclear export of p53 by poly(ADP-ribosyl)ation.

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.

Combined effects of polymorphisms of DNA-repair protein genes and metabolic enzyme genes on the risk of cholangiocarcinoma.

OBJECTIVE: Although Opisthorchis viverrini is a risk factor for cholangiocarcinoma, not all the infected individuals develop cholangiocarcinoma. We investigated whether the base excision repair enzyme gene polymorphisms with differentiated repair capacities of inflammation-related deoxyribonucleic acid damage may play a key role and such possible effects from those genes may be increased or diminished in co-existence of polymorphisms of metabolic enzymes, including glutathione-S-transferases mu 1 and glutathione-S-transferases θ1. METHODS: We genotyped five non-synonymous single-nucleotide polymorphisms of three genes, including the human homolog of the 8-oxoguanine glycosylase 1 Ser326Cys, X-ray repair cross-complementing protein 1 Arg194Trp, Arg280His and Arg399Gln and poly (adenosine diphosphate ribose) polymerase 1 Val762Ala in 87-94 matched case-control pairs, and examined relations between those polymorphisms and the risk of cholangiocarcinoma. RESULTS: Any single polymorphism did not have a measurable association with the risk of cholangiocarcinoma. However, when considering glutathione-S-transferases mu 1 polymorphism together, the human homolog of the 8-oxoguanine glycosylase 1 codon 326 polymorphism was related to the decreased risk; odds ratios were 1.00 (reference), 0.06 (95% confidence interval 0.01-0.53), 0.06 (0.01-0.54) and 0.14 (0.02-1.08) for persons with human homolog of the 8-oxoguanine glycosylase 1 Ser/Ser and glutathione-S-transferases mu 1 wild, ones with Ser/Ser and glutathione-S-transferases mu 1 null, ones with Ser/Cys or Cys/Cys and glutathione-S-transferases mu 1 wild and ones with Ser/Cys or Cys/Cys and glutathione-S-transferases mu 1 null, respectively (P for interaction <0.01). Further adjustment for the presence of anti-Opisthorchis viverrini antibody, smoking and alcohol drinking did not change the decreased risk. Other combinations of deoxyribonucleic acid-repair gene polymorphism and glutathione-S-transferases were not associated with the risk of cholangiocarcinoma. CONCLUSIONS: The present findings suggested that decreased capacity of deoxyribonucleic acid-repair gene, human homolog of the 8-oxoguanine glycosylase 1, may be related to decreased risk if much damaged cells die before malignant transformation.

NAD+ Consuming Enzymes: Involvement in Therapies and Prevention of Human Diseases.

Neuroprotection is one of the hot topics in medicine. Alzheimer's disease, amyotrophic lateral sclerosis, retinal pigment epithelial (RPE) degeneration, and axonal degeneration have been studied for the involvement of NAD depletion. Localized NAD+ depletion could lead to overactivation and crowding of local NAD+ salvage pathways. It has been stated that NAD+ depletion caused by PARPs and PAR cycling has been related to metabolic diseases and cancer. Additionally, it is now acknowledged that SARM1 dependent NAD+ depletion causes axon degeneration. New targeted therapeutics, such as SARM1 inhibitors, and NAD+ salvage drugs will help alleviate the dysfunctions affecting cell life and death in neurodegeneration as well as in metabolic diseases and cancer.

An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo.

PolyADP-ribosylation is a posttranslational modification of proteins that results from enzymatic synthesis of poly(ADP-ribose) with NAD+ as the substrate. A unique characteristic of polyADP-ribosylation is that the poly(ADP-ribose) chain can have 200 or more ADP-ribose residues in branched patterns, and the presence and variety of these chains can have substantive effects on protein function. To understand how polyADP-ribosylation affects biological processes, it is important to know the physiological level of poly(ADP-ribose) in cells. Under normal cell physiological conditions and in the absence of any exogenous DNA damaging agents, we found that the concentration of poly(ADP-ribose) in HeLa cells is approximately 0.04 pmol (25 pg)/106 cells, as measured with a double-antibody sandwich, enzyme-linked immunosorbent assay protocol that avoids artificial activation of PARP1 during cell lysis. Notably, this system demonstrated that the poly(ADP-ribose) level peaks in S phase and that the average cellular turnover of a single poly(ADP-ribose) is less than 40 s.

Polymorphism of genes encoding drug-metabolizing and inflammation-related enzymes for susceptibility to cholangiocarcinoma in Thailand.

BACKGROUND: Cholangiocarcinoma (CCA) is an intractable cancer, and its incidence in northeastern Thailand is the highest worldwide. Infection with the liver fluke Opisthorchis viverrini (OV) has been associated with CCA risk. However, animal experiments have suggested that OV alone does not induce CCA, but its combination with a chemical carcinogen like nitrosamine can cause experimentally induced CCA in hamsters. Therefore, in humans, other environmental and genetic factors may also be involved. AIM: To examine relations between risk for CCA and genetic polymorphisms in carcinogen-metabolizing and inflammation-related genes. METHODS: This hospital-based case-control study enrolled 95 case-control pairs matched by age (± 5 years) and sex. We examined relations between risk for CCA and genetic polymorphisms in carcinogen-metabolizing and inflammation-related genes, serum anti-OV, alcohol consumption, and smoking. Polymorphisms of CYP2E1, IL-6 (-174 and -634), IL-10 (-819), and NF-κB (-94) and their co-occurrence with polymorphisms in the drug-metabolizing enzyme gene GSTT1 or GSTM1 were also analyzed. RESULTS: Although CCA risk was not significantly associated with any single polymorphism, persons with the GSTT1 wild-type and CYP2E1 c1/c2 + c2/c2 genotype had an increased risk (OR = 3.33, 95%CI: 1.23-9.00) as compared with persons having the GSTT1 wild-type and CYP2E1 c1/c1 wild genotype. The presence of anti-OV in serum was associated with a 7- to 11-fold increased risk, and smoking level was related to an OR of 1.5-1.8 in multivariable analyses adjusted for each of the seven genetic polymorphisms. CONCLUSION: In addition to infection with OV, gene-gene interactions may be considered as one of the risk factors for CCA development.

Novel pathway of centrosome amplification that does not require DNA lesions.

Centrosome amplification (also known as centrosome overduplication) is common in cancer cells and can be induced by DNA damaging agents. However, the mechanism and significance of centrosome amplification during carcinogenesis or after DNA damage are not clear. Previously, we showed that centrosome amplification could be induced by 3-aminobenzamide (3-AB), an inhibitor of poly(ADP-ribose) polymerases (PARPs) in mouse embryonic fibroblasts. In this paper, we determined if the effect of 3-AB on centrosome amplification was dependent on DNA damage in CHO-K1 cells. We used the well-known mutagen/carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Ten micromolar MNNG and 10 mM 3-AB induced significant centrosome amplification in 18.1 ± 1.1% and 19.4 ± 1.8% of CHO-K1 cells, respectively, compared to 7.0 ± 0.5% of untreated CHO-K1 cells. AG14361, another potent inhibitor of PARPs, also induced centrosome amplification. We then used γ-H2AX analysis and alkaline comet assays to show that 10 µM MNNG induced a significant number of DNA lesions and cell cycle arrest at the G(2) /M phase. However, 10 mM 3-AB neither induced DNA lesions nor altered cell cycle progression. In the umu test, 10 µM MNNG was mutagenic, but 10 mM 3-AB was not. In addition, 10 µM MNNG induced significant accumulation of ataxia telangiectasia mutated protein in the nuclei, but 10 mM 3-AB did not. Thus, we found no association between apparent DNA lesions and centrosome amplification after 3-AB treatment. Therefore, we propose the presence of a novel pathway for centrosome amplification that does not necessarily require DNA lesions but involves regulation of epigenetic changes or post-translational modifications including polyADP-ribosylation.

PRKAR1A overexpression is associated with increased ECPKA autoantibody in liver fluke-associated cholangiocarcinoma: application for assessment of the risk group.

Cholangiocarcinoma (CCA) associated with Opisthorchis viverrini (Ov) chronic infection is the most frequent primary liver cancer in Thailand, and current approaches to early diagnosis and curative treatments are largely disappointing. We hypothesize a role for protein kinase A (PKA) in Ov-induced CCA. First, we studied the PKA isozyme switching in the liver from the hamster CCA model using quantitative (q) PCR, in situ hybridization, and immunohistochemical and western blot analysis. Second, the presence of extracellular PKA (ECPKA) in CCA cell lines and their conditioned media was demonstrated by western blot and PKA activity assay. Third, we determined the association between PRKAR1A expression and serum ECPKA autoantibody in patients with CCA by ELISA. We demonstrated that an increased PRKAR1A expression is restricted to the biliary cells starting from week 1, with remarkable up-regulation when CCA has completely developed by week 24. The switching of the PKA regulatory subunit isoforms from PRKAR2B/PKAII to PRKAR1A/PKAI is significantly associated with cholangiocyte proliferation. Further, we observed that human CCA cell lines express PRKAR1A but not PRKAR2B and excrete ECPKA. Finally, ECPKA autoantibodies are detected in serum of patients with CCA, adenocarcinoma, and Ov infection with periductal fibrosis, but not from Ov-infected subjects without periductal fibrosis lesion and healthy controls. We conclude that PKA isozyme switching and the PRKAR1A/PKAI pathway might contribute to the induction of cholangiocyte transformation and proliferation in Ov-induced CCA. Overexpression of PRKAR1A leads to secretion of ECPKA which is associated with serum autoantibody that may constitute a biomarker for human CCA genesis.

PRKAR1A is overexpressed and represents a possible therapeutic target in human cholangiocarcinoma.

The protein kinase A regulatory subunit 1 alpha (PRKAR1A/PKAI) pathway is overexpressed in varieties of tumors and cancer cell lines including cholangiocarcinoma (CCA), although its role in CCA growth modulation is unclear. In our study, we evaluated the effect of PRKAR1A/PKAI targeting on CCA cell proliferation. Real-time PCR demonstrated an increased mRNA expression of PRKAR1A/PKAI, whereas protein kinase A regulatory subunit 2 beta (PRKAR2B/PKAII) was downregulated in human CCA tissues and CCA cell lines. Immunohistochemistry of human CCA tissues revealed increased PRKAR1A with decreased PRKAR2B protein expression. Moreover, CCA cell lines showed abundantly expressed PRKAR1A, while lacking PRKAR2B expression. Silencing PRKAR1A expression induced growth inhibition and apoptosis of CCA cells, with an associated decrease in mitogen-activated protein kinases, PI3K/Akt, JAK/STAT and Wnt/ß-catenin pathway signaling. The inhibition of PKA using a PKA inhibitor and cAMP analogs also led to a significant cell growth inhibition. In conclusion, our study reports the overexpression as well as molecular mechanisms by which PRKAR1A/PKA regulates human CCA cell growth. Importantly, abrogation of gene expression caused significant CCA cell growth inhibition, oncogenic signaling and coupled apoptosis induction, suggesting PRKAR1A's potential as a drug target for CCA therapy.

Myristoylated alanine-rich C kinase substrate phosphorylation promotes cholangiocarcinoma cell migration and metastasis via the protein kinase C-dependent pathway.

Myristoylated alanine-rich C kinase substrate (MARCKS), a substrate of protein kinase C (PKC) has been suggested to be implicated in cell adhesion, secretion, and motility through the regulation of the actin cytoskeletal structure. The quantitative real-time-polymerase chain reaction analysis revealed that MARCKS is significantly overexpressed in Opisthorchis viverrini-associated cholangiocarcinoma (CCA) (P = 0.001) in a hamster model, which correlated with the results of mRNA in situ hybridization. An immunohistochemical analysis of 60 CCA patients revealed a significant increase of MARCKS expression. Moreover, the log-rank analysis indicated that CCA patients with a high MARCKS expression have significantly shorter survival times than those with a low MARCKS expression (P = 0.02). This study investigated whether MARCKS overexpression is associated with CCA metastasis. Using a confocal microscopic analysis of CCA cell lines that had been stimulated with the PKC activator, 12-0-tetradecanoyl phorbol-13-acetate (TPA), MARCKS was found to be translocated from the plasma membrane to the perinuclear area. In addition, phosphorylated MARCKS (pMARCKS) became highly concentrated in the perinuclear area. Moreover, an adhesion assay demonstrated that the exogenous overexpression of MARCKS remarkably promoted cell attachment. Interestingly, after TPA stimulation, the CCA cell line-depleted MARCKS showed a decrease in migration and invasion activity. It can be concluded that in non-stimulation, MARCKS promotes cell attachment to the extracellular matrix. After TPA stimulation, PKC phosphorylates MARCKS leading to cell migration or invasion. Taken together, the results of this study reveal a prominent role for MARCKS as one of the key players in the migration of CCA cells and suggest that cycling between MARCKS and pMARCKS can regulate the metastasis of biliary cancer cells.

Drosophila melanogaster as a model for understanding polyADP-ribosylation.

PolyADP-ribosylation is a post-translational modification which is involved in various physiological processes including maintenance of genome stability through DNA repair, regulation of transcription, and development. This process is also involved in pathological events such as cell death. Here, we review the effect of polyADP-ribosylation in signal transduction pathways in Drosophila melanogaster system. It is hoped that such an insight paves the way to develop therapeutics for human diseases.

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1.

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27(Kip1) gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependent on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27(Kip1) gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27(Kip1) gene expression.

Single-particle analysis of full-length human poly(ADP-ribose) polymerase 1.

PolyADP-ribosylation (PARylation) is a posttranslational modification that is involved in the various cellular functions including DNA repair, genomic stability, and transcriptional regulation. PARylation is catalyzed by the poly(ADP-ribose) polymerase (PARP) family proteins, which mainly recognize damaged DNA and initiate repair processes. PARP inhibitors are expected to be novel anticancer drugs for breast and ovarian cancers having mutation in BRCA tumor suppressor genes. However the structure of intact (full-length) PARP is not yet known. We have produced and purified the full-length human PARP1 (h-PARP1), which is the major family member of PARPs, and analyzed it with single particle electron microscopy. The electron microscopic images and the reconstructed 3D density map revealed a dimeric configuration of the h-PARP1, in which two ring-shaped subunits are associated with two-fold symmetry. Although the PARP1 is hypothesized to form a dimer on damaged DNA, the quaternary structure of this protein is still controversial. The present result would provide the first structural evidence of the dimeric structure of PARP1.

Effects of radiation based on whole-body irradiation in HTLV-1-infected mice.

Adult T-cell leukemia is one of the life-threatening diseases that occur in individuals infected with human T-cell leukemia virus type 1 (HTLV-1). Clinical trials of hematopoietic stem cell transplantation therapy are being performed in addition to chemotherapy; however, neither is satisfactory. As a pretreatment for transplantation, anticancer drugs or whole-body irradiation is used to decrease the number of HTLV-1-infected cells, but there are numerous side effects. Therefore, in the present study, using a mouse model of HTLV-1 infection, the long-term survival and number of infected cells in the reservoir organ were investigated in order to determine the effect of γ-irradiation on HTLV-1-infected mice in vivo. There was no improvement in the survival period following γ-irradiation in the γ-irradiated group after HTLV-1 infection when compared with the HTLV-1-infected group. It was also found that the incidence of splenomegaly was ≥80% in the HTLV-1-infected and γ-irradiated group, which was significantly higher than that in the HTLV-1-infected mice. The tissue morphology in the spleen became non-uniform because of γ-rays. Importantly, the number of infected cells in the spleen was increased 4.1-fold in the HTLV-1-infected and γ-irradiated mice compared with that in the HTLV-1-infected mice. Careful consideration might be necessary when using whole-body irradiation in patients with HTLV-1 infection.