Search

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.

Alcohol dehydrogenase (ADH) is important for preventing alcohol toxicity and developmental disorders, and may be involved in other diseases including neurodegenerative diseases. We found that the major acceptor protein of polyADP-ribosylation in a model organism of neurodegeneration using a Drosophila melanogaster mutant lacking poly(ADP-ribose) glycohydrolase, was ADH. Thus we postulated that human ADH activity might be regulated by polyADP-ribosylation, a post-translational modification. The radioactivity of [32P]NAD+ was incorporated into human ADH1 by human poly(ADP-ribose) polymerase 1 in vitro, but was not incorporated when heat-inactivated PARP1 or a PARP inhibitor, 3-aminobenzamide, was used. The incorporated radioactivity was not released from ADH1 protein in the presence of excess amount of ADP-ribose or poly(ADP-ribose) as competitors. However, it was released by incubation with 1 M neutral NH2OH or 0.1 N NaOH, but was not with 0.1 N HCl, suggesting the bond between ADH1 and poly(ADP-ribose) is an ester linkage. When HepG2 cells, a human hepatoma cell line, were cultured in the presence of another PARP inhibitor, olaparib, ADH activity of the cell was significantly increased. These results suggest that polyADP-ribosylation could regulate ADH activity in vivo and might be involved in neurodegeneration.

The characteristics of two monoclonal antibodies to poly(ADP-Rib) and the various structures of poly(ADP-Rib) recognized by these monoclonal antibodies have been examined. One antibody, IgG monoclonal antibody 10H, reacted with most parts of poly (ADP-Rib) molecules, and its binding was only slightly inhibited by Ado(P)-Rib-P, a monomer unit of the polymer. On hydrolysis of poly(ADP-Rib) protected by the antibody hydrolytic products, such as Ado(P)-Rib-P and Ado(P)-Rib(P)-Rib-P, were almost the same as those from control poly(ADP-Rib). The other antibody, IgM monoclonal antibody 16B, recognized only some parts of poly(ADP-Rib) molecules of larger size, and its binding was not inhibited by Ado(P)-Rib-P. Branched portions were considerably concentrated in parts protected by this antibody. These two monoclonal antibodies are suggested to recognize different structures of poly(ADP-Rib): possibly IgG antibody 10H recognizes linear portions whereas IgM antibody 16B recognizes branched portions of the polymer. These monoclonal antibodies should be useful in studies on the structures, including unknown structures and the locations of these structures in poly(ADP-Rib).

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.

Adult T-cell leukemia and human T-cell leukemia virus type 1 (HTLV-1) - associated myelopathy/tropical spastic paraparesis, which develop after HTLV-1 infection, are difficult to cure. In particular, the mode of HTLV-1 propagation is not well understood. Poly (ADP-ribose) polymerase-1 is reported to be a co-activator of HTLV-1 Tax protein; however, the effects of polyADP-ribosylation on infectivity of HTLV-1 have not been fully clarified. We studied the effects of a PARP inhibitor on two modes of HTLV-1 transmission: through cell adhesion between MT-2 cells (an HTLV-1-infected cell line) and uninfected cells and through virus particles produced by HTLV-1-producing c77 cells. Although the PARP inhibitor decreased HTLV-1 infection through cell adhesion, it increased HTLV-1 infection through virion production and caused apoptosis of HTLV-1-infected cells. Thus, careful consideration is required for clinical application of PARP inhibitors in HTLV-1 patients.

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.

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 (p0.0001). 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: p0.0001, 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.

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.

P5, one of the protein disulphide isomerase (PDI) family members, catalyses disulphide bond formation in proteins and exhibits molecular chaperone and calcium binding activities in vitro, whereas its physiological significance remains controversial. Recently, we have reported that P5 localizes not only in the ER but also in mitochondria, although it remains unclear so far about its physiological significance(s) of its dual localization. Here we report that H(2)O(2)- or rotenone-induced cell death is suppressed in MTS-P5 cells, which stably express P5 in mitochondria. H(2)O(2)-induced cell death in Saos-2 cells occurred, in large part, through caspase-independent and poly(ADP-ribose) polymerase (PARP)-dependent manner. In MTS-P5 cells challenged with H(2)O(2) treatment, PARP was still activated, whereas release of cytochrome c or apoptosis-inducing factor and intramitochondrial superoxide generation were suppressed. We also found that mitochondrial P5 was in close contact with citrate synthase and maintained large parts of its activity under H(2)O(2) exposure. These results suggest that mitochondrial P5 may upregulate tricarboxylic acid cycle and possibly, other intramitochondrial metabolism.

Myristoylated alanine rich C kinase substrate (MARCKS) has been implicated in PKC-mediated membrane-cytoskeleton alterations that underlie lipopolysaccharide (LPS)-induced macrophage responses. MARCKS is postulated to be involved in inflammation-associated CCA based on its overexpression in cholangiocarcinoma (CCA) and inflammatory cells. The aims of this study were to investigate localization patterns of MARCKS in hamster and human tissue during cholangiocarcinogenesis and to examine the involvement of MARCKS in inflammation. MARCKS protein expression was found prominently in inflammatory cells of Opisthorchis viverrini -treated as well as O. viverrini plus N -nitrosodimethylamine (NDMA)-treated hamsters from week 2 to week 3 of treatment. The positive signal decreased during week 4 to week 12, then increased again at week 26 when CCA developed. At the last time point the expression of MARCKS was observed in both cancer and inflammatory cells. MARCKS protein expression was also found in inflammatory cells, including macrophages in human CCA tissues. O. viverrini excretory/secretory products or worm antigen induced MARCKS mRNA and protein expression in a dose- and time-dependent manner in the human U937 macrophage cell line. The relative mRNA expression of MARCKS in white blood cells of O. viverrini -infected patients was significantly higher than in healthy subjects ( P = 0.02). Thus, MARCKS is significantly expressed in macrophages and plays a role in inflammation-related CCA induced by O. viverrini .

Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia and HTLV-1-associated myelo-pathy/tropical spastic paraparesis. HTLV-1 is mainly transmitted through blood transfusion and breastfeeding, but viral proliferation in the body in vivo shortly after transmission is not well understood. To investigate whether the route of infection influences the early stages of viral proliferation, we inoculated BALB/c mice with MT-2 cells, an HTLV-1-producing human T-cell line, via different routes, and evaluated the proviral load and humoral immune responses. One month after infection, the provirus was detected in most organs of the mice infected intraperitoneally, and substantial proviral loads were detected in the peripheral blood and secondary lymphoid organs. In contrast, the mice infected intravenously and orally showed low proviral loads, and the provirus distribution was limited to the spinal cord among the intravenously inoculated mice and to the liver among the perorally inoculated mice. Mice infected intraperitoneally also exhibited higher interleukin-2 production than the mice infected intravenously or orally, or than the uninfected control mice, while anti-HTLV-1 antibody titers were comparable between the mice infected intraperitoneally and intravenously. These results demonstrate that the route of primary HTLV-1 infection influences the establishment of HTLV-1-infected cell proliferation and the cell reservoir in mice.

Human T-cell leukemia virus type 1 (HTLV-1) infection is involved in the development of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. A high HTLV-1 proviral load in circulating lymphocytes of HTLV-1 carriers is a risk factor for HTLV-1-related diseases. The virus-cell interaction is linked to viral tropism and pathogenesis. Characterization of the factors that affect HTLV-1 infection is important for preventing HTLV-1 infection. HTLV-1 virions are believed to be weakly infectious under cell culture conditions; however, we found that the treatment of HTLV-1 virions with microbial neuraminidase, an enzyme catalyzing the removal of sialic acid residues from various glycoconjugates, enhanced the number of proviral DNAs in infected cells in a dose-dependent manner. Neuraminidase treatment of virions, but not target cells, enhanced viral binding and entry into cells and viral infectivity; treatment of target cells prior to infection had no effect. Moreover, the number of HTLV-1-mediated syncytia was higher in the presence of neuraminidase. Our results suggest a possible contribution of microbial agents carrying neuraminidase activity to HTLV-1 pathogenesis.

Human T-cell lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory disorders. Aberrant activation of nuclear factor-κB (NF-κB) has been linked to HTLV-1 pathogenesis and to various kinds of cancers, including adult T-cell leukemia. NF-κB-inducing kinase (NIK) is critical for non-canonical activation of NF-κB and for the development of lymphoid organs. HTLV-1 activates NF-κB by the non-canonical pathway, but examination of the role of NIK in proliferation of HTLV-1-infected cells in vivo has been hindered by lack of a suitable animal model. Alymphoplasia (aly/aly) mice bear a mutation of NIK, resulting in defects in the development of lymphoid organs and severe deficiencies in both humoral and cell-mediated immunity. In the present study we therefore used a mouse model of HTLV-1 infection with aly/aly mice. The number of HTLV-1-infected cells in the reservoir organs in aly/aly mice was significantly smaller than in the control group 1 month after infection. In addition, aly/aly mice did not maintain provirus for 1 year and antibodies against HTLV-1 were undetectable. These results demonstrate that the absence of functional NIK impairs primary HTLV-1 proliferation and abolishes the maintenance of provirus. Interestingly, clonal proliferation of HTLV-1-infected mouse cells was not detected in aly/aly mice, which is consistent with the lack of HTLV-1 persistence. These observations imply that the clonal proliferation of HTLV-1-infected cells in secondary lymphoid organs might be important for HTLV-1 persistence. (Cancer Sci 2008; 99: 872–878)

The polyADP-ribosylation reaction results in a unique post-translational modification involved in various cellular processes and conditions, including DNA repair, transcriptional control, genomic stability, cell death and transformation. The existence of 17 members of the poly(ADP-ribose) polymerase (PARP) family has so far been documented, with overlapping functional consequences. PARP-1 is known to be involved in DNA base excision repair and this explains the susceptibility spectrum of PARP-1 knockout animals to genotoxic carcinogens. The fact that centrosome amplification is induced by a non-genotoxic inhibitor of PARP and in PARP-1 knockout mouse cells, is in line with aneuploidy, which is frequent in cancers. Genetically engineered animal models have revealed that PARP-1 and VPARP impact carcinogenesis. Furthermore, accumulating experimental evidence supports the utility of PARP and PARG inhibitors in cancer therapy and several clinical trials are now ongoing. Increasing NAD(+) levels by pharmacological supplementation with niacin has also been found to exert preventive effects against cancer. In the present review, recent research progress on polyADP-ribosylation related to neoplasia is summarized and discussed.

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.

In higher eukaryotes and plants poly-ADP-ribose polymerases (PARPs) are involved in chemical and oxidative damage. PARP proteins are shown to confer resistance to reactive oxygen species and to oxidative stress. PARP enzymes and plant PARP-domain proteins show to exert a control of chromatin, nucleosomes, gene silencing and epigenetic regulation by transcription factor complexes. In addition, PARP activity regulates NAD pool. Synthesis and stability of pADR polymer seems to influence cell survival or death. Plant PARP-domain proteins have been shown able to regulate the formation and activity of transcription factor complexes involved in the activation of flavonoid biosynthesis genes, supporting anthocyanin accumulation, in stressed and non-stressed plants.

Infection by Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. However, the mechanism by which the parasite is involved in carcinogenesis is not clear. In addition to the direct damage of the bile duct epithelium via direct contact with O. viverrini, the excretory/secretory (ES) product(s) released from the parasites may play important roles in this process. We therefore investigated the responses of a fibroblast cell line, NIH-3T3, to ES product(s) released from O. viverrini by using a non-contact co-culture technique. In this culture system, the parasites in the upper chamber had no direct contact with the NIH-3T3 cells in the lower chamber of the culture plate. The results indicated a marked increase in NIH-3T3 cell proliferation in the non-contact co-culture condition with either 0% or 10% calf serum in the medium compared with that without parasites. ES product(s) increased cell proliferation by stimulating the expression of phosphorylated retinoblastoma (pRB) and cyclin D1, the key proteins in driving cells through the G1/S transition point of the cell cycle. This led to the induction of cells going into the S-phase of the cell cycle. ES product(s) also changed the morphology of NIH-3T3 cells to a refractive and narrow shape, which allowed the cells to proliferate in the limited culture area. For the first time, we have been able to demonstrate increased cell proliferation induced by the ES product(s) from O. viverrini; this finding may clarify how O. viverrini ES product(s) affect human bile duct epithelium during cholangiocarcinogenesis.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr appears to make a substantial contribution to the replication of HIV-1 in established T cell lines when HIV-1 is present at very low multiplicities of infection. However, the role of Vpr in viral replication in primary CD4+ T cells remains to be clarified. In this study, we generated a panel of viruses that encoded mutant forms of Vpr that lacked either the ability to accumulate in the nucleus and induce G2 arrest or the ability to induce apoptosis, which has been shown to occur independently of G2 arrest of the cell cycle. We demonstrate here that the nuclear localization of Vpr and consequent G2 arrest but not the induction of apoptosis by Vpr are important for viral replication in primary CD4+ T cells at both high and low multiplicities of infection. Viruses that encoded mutant forms of Vpr that failed to be imported into the nucleus in the presence of cytoplasmic extracts from primary CD4+ T cells in an in vitro nuclear import assay replicated at drastically reduced rates. Thus, Vpr might be a key regulator of the viral nuclear import process during infection in primary CD4+ T cells. By contrast, a mutant form of Vpr that exhibited diffuse cytosolic staining exclusively in an immunofluorescence assay of HeLa cells and was not imported into nucleus by the cytosol from HeLa cells was effectively imported into the nucleus by cytosol from primary CD4+ T cells. This Vpr mutant virus replicated well in primary CD4+ T cells, indicating that cellular factors in primary CD4+ T cells are indispensable for the accumulation of Vpr in the nucleus and, thus, for viral replication. Our results suggest that the nuclear import of Vpr might be a good target in efforts to block the early stages of replication of HIV-1.

Adult T-cell leukemia (ATL) occurs after a long latent period of persistent infection by human T-cell leukemia virus type 1 (HTLV-1). However, the mechanism of oncogenesis by HTLV-1 remains to be clarified. It was reported that the incidence curve of ATL versus age was consistent with a multistage carcinogenesis model. Although HTLV-1 is an oncogenic retrovirus, a mechanism of carcinogenesis in ATL by insertional mutagenesis as one step during multistage carcinogenesis has not been considered thus far, because the exact integration sites on the chromosome have not been analyzed. Here we determined the precise HTLV-1 integration sites on the human chromosome, by taking advantage of the recently available human genome database. We isolated 25 integration sites of HTLV-1 from 23 cases of ATL. Interestingly, 13 (52%) of the integration sites were within genes, a rate significantly higher than that expected in the case of random integration (P = 0.043, chi(2) test). These results suggest that preferential integration into genes at the first infection is a characteristic of HTLV-1. However considering that some of the genes are related to the regulation of cell growth, the integration of HTLV-1 into or near growth-related genes might contribute to the clonal selection of HTLV-1-infected cells during multistage carcinogenesis of ATL.

Purpose: Cancer diagnostics and therapeutics are often based on clinically relevant markers that are expressed specifically in a malignant tissue at levels higher than in normal tissue. We examined potential markers for papillary thyroid carcinoma (PTC) by monitoring PTC-specific gene expression using cDNA microarray. Experimental Design: Gene expression profiles for PTC tissue, normal thyroid tissue, and healthy peripheral blood cells were compared by use of a human 4000-gene cDNA microarray. Protein expressions of the up-regulated genes in PTC were examined in thyroid tissues by immunohistochemistry. Results: Sixty-four genes were overexpressed in PTC tissue relative to normal thyroid tissue and healthy peripheral blood cells. The genes that were up-regulated in PTC were involved in cell cycle regulation, DNA damage response, angiogenesis, and oncogenesis. Among these genes, basic fibroblast growth factor and platelet-derived growth factor were identified by immunochemical methods as proteins that are specifically expressed at high levels in thyroid neoplasms. Basic fibroblast growth factor, which has been identified as a biomarker for PTC, was overexpressed in 54% of PTC cases, 67% of follicular thyroid carcinomas, and 36% of benign thyroid neoplasms. Platelet-derived growth factor was overexpressed in 81% of PTC cases and 100% of follicular carcinomas, but was immunonegative in normal thyroid tissues and benign thyroid neoplasms. Conclusions: Platelet-derived growth factor may be a potential biomarker for PTC and follicular carcinoma. Expression profile analysis using a microarray followed by immunohistochemical study can be used to facilitate the development of molecular biomarkers for cancer.

Centrosomes play important roles in cell polarity, regulation of cell cycle and chromosomal stability. Centrosome abnormality is frequently found in many cancers and contributes to chromosomal instability (including aneuploidy, tetraploidy, and/or micronuclei) in daughter cells through the assembly of multipolar or monopolar spindles during mitosis. It has recently been reported that loss of tumor suppressor genes or overexpression of oncogenes causes centrosome hyperamplification. Amplification and overexpression of the MYCN oncogene is found in a subgroup of neuroblastomas. In this study, we examined whether overexpression of MYCN causes centrosome hyperamplification in neuroblastoma cells. We show that ectopic expression of MYCN alone in a neuroblastoma cell line did not cause centrosome hyperamplification. However, centrosome hyperamplification and micronuclei formation were seen in these cells after DNA damage. These findings suggest that overexpression of MYCN abrogates the regulation of the centrosome cycle after DNA damage.

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked with HTLV-1-associated diseases. HTLV-1 proviral load is higher in persons with adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers. However there are little data available on the factors controlling HTLV-1 proviral load in carriers. To study the effect of genetic background on HTLV-1 proviral load, we employed a mouse model of HTLV-1 infection that we had established. Here we analyzed nine strains of mice and found there is a great variation of proviral load among mouse strains that is not necessarily dependent on major histocompatibility complex. The antibody response is also different among these strains. To our knowledge, this is the first demonstration of the importance of the genetic background other than major histocompatibility complex controlling the HTLV-1 proviral load.

Posttranslational modification plays important roles in a range of cellular functions. Poly(ADP-ribosyl)ation influences DNA repair, transcription, centrosome duplication, and chromosome stability. Poly(ADP-ribose) attached to acceptor proteins should be properly hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). However the subcellular localization and the role of PARG have not been well characterized. Here, we transiently expressed GFP- or Myc-tagged human PARG in mammalian cells and revealed that the subcellular distribution of human PARG changes dramatically during the cell cycle. GFP-hPARG is found almost exclusively in the nucleus during interphase. During mitosis, most GFP-hPARG protein localizes to the cytoplasm and hardly any GFP-hPARG protein is found associated with the chromosomes. Furthermore, we found that GFP-hPARG localizes to the centrosomes during mitosis. Our findings suggest that shuttling of PARG between nucleus and cytoplasm and proper control of poly(ADP-ribose) metabolism throughout the cell cycle may play an important role in regulating cell cycle progression and centrosome duplication.

The regulatory mechanism of centrosome function is crucial to the accurate transmission of chromosomes to the daughter cells in mitosis. Recent findings on the posttranslational modifications of many centrosomal proteins led us to speculate that these modifications might be involved in centrosome behavior. Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes poly(ADP-ribosyl)ation to various proteins. We show here that PARP-1 localizes to centrosomes and catalyzes poly(ADP-ribosyl)ation of centrosomal proteins. Moreover, centrosome hyperamplification is frequently observed with PARP inhibitor, as well as in PARP-1-null cells. Thus, it is possible that chromosomal instability known in PARP-1-null cells can be attributed to the centrosomal dysfunction. P53 tumor suppressor protein has been also shown to be localized at centrosomes and to be involved in the regulation of centrosome duplication and monitoring of the chromosomal stability. We found that centrosomal p53 is poly(ADP-ribosyl)ated in vivo and centrosomal PARP-1 directly catalyzes poly(ADP-ribosyl)ation of p53 in vitro. These results indicate that PARP-1 and PARP-1-mediated poly(ADP-ribosyl)ation of centrosomal proteins are involved in the regulation of centrosome function.

Human T-cell leukemia virus type 1 (HTLV-1) is an etiologic agent of adult T-cell leukemia / lymphoma and other HTLV-1-associated diseases. However, the interaction between HTLV-1 and T cells in the pathogenesis of these diseases is poorly understood. Mouse cells have been reported to be resistant to cell-free HTLV-1 infection. However, we recently reported that HTLV-1 DNA could be observed 24 h after cell-free HTLV-1 infection of mouse cell lines. To understand HTLV-1 replication in these cells in detail, we concentrated the virus produced from c77 feline kidney cell line and established an efficient infection system. The amounts of adsorption of HTLV-1 are larger in mouse T cell lines, EL4 and RLm1, than those in human T cell lines, Molt4 and HUT78, and are similar to that in human kidney cell line, 293T. Unexpectedly, however, the amounts of entry of HTLV-1 are about 10-fold larger in the two mouse cell lines than those in the three human cell lines employed. Moreover, viral DNA was detectable from 1 h in EL4 and RLm1 cells, but only from 2 - 3 h in 293T, Molt4 and HUT78 cells. However, the amount of viral DNA in EL4 cells became smaller than that in Molt4 cells. HTLV-1 expression could be detected until day 1 - 2 in RLm1 and EL4 cells, and until day 4 in Molt4 cells. Our results suggest that mouse cell experiments would give useful information to dissect the early steps of cell-free HTLV-1 infection.

Postsurgical recurrence at distant sites frequently occurs in pathological tumor stage 2 (pT(2)) carcinoma of the gallbladder even though the carcinoma is limited to the gallbladder wall. Little is known, however, about the molecular events leading to its development and progression. A large body of evidence suggests that cyclooxygenase-2 (COX-2) is up-regulated in carcinoma tissues and plays roles in promoting cell-proliferation, growth and metastasis of carcinoma cells. In the present study, immunohistochemistry was performed to determine the expression levels of COX-2 in the subserosal layer of 33 cases of pT(2) gallbladder carcinoma in which curative resections had been performed and to determine the correlations of the expression levels of COX-2 with mode of recurrence and postsurgical survival. Immunostaining of COX-2 in the epithelia was recognized in more than 80% of normal epithelia, noncancerous pathological lesions of the gallbladder except for intestinal metaplasia and pT(1-4) carcinoma specimens. Intense staining was observed in large percentages of hyperplastic lesions (65%), pT(2) carcinoma specimens (76%) and pT(3) and pT(4) carcinoma specimens (64%) compared to the percentages of normal epithelia and other pathological lesions (0-25%). Intense staining was also observed in the adjacent stroma in pT(2) carcinoma specimens (33%) and in those in pT(3) and pT(4) carcinoma specimens (43%) but only in small percentages of the stroma adjacent to normal epithelia and pathological lesions (0-8%). In situ hybridization confirmed the existence of COX-2 mRNA in both the cancerous epithelia and adjacent stroma of pT(2)-pT(4) carcinomas. In 33 cases of pT(2) carcinoma, distant recurrence, i.e., liver metastasis, was seen in 3 of 9 cases of pT(2) carcinoma (33%, P0.05) with intense stromal staining in the subserosal layer and in 1 of 24 cases (4%) without intense staining, whereas no significant correlation was found between parameters of pathological malignancies (histological grade, lymphatic permeation, venous permeation and lymph node metastasis) and the intensity of stromal staining in the subserosal layer. The postsurgical survival outcome was significantly poorer in the former than in the latter (p = 0.010). In pT(2) gallbladder carcinoma, upregulation of COX-2 in the stroma adjacent to the cancerous epithelia in the subserosal layer correlates with the aggressiveness of the disease, such as the tendency to form distant recurrences. This phenotype may serve as a unique biological feature associated with the malignant behavior of pT(2) gallbladder carcinoma.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for adult T-cell leukemia and HTLV-1-associated myelopathy / tropical spastic paraparesis. Recently we infected newborn mice by inoculating HTLV-1-producing human cells, and found that T-cells, B-cells and granulocytes were infected in vivo. To understand the mechanism of viral-cell interaction and the pathogenesis of HTLV-1 using the mouse model, it is important to clarify the cellular tropism using a cell-free HTLV-1 transmission system. We employed a highly transmissible cell-free HTLV-1 produced by a feline kidney cell line, c77, and studied the susceptibility of 9 kinds of mouse cell lines, EL4, RLm1, CTLL-2, J774.1, DA-1, Ba / F3, WEHI-3, NIH3T3 and B1, and two kinds of human cell lines, Molt-4 and Hut78. HTLV-1 proviral sequence was found by PCR in all 9 mouse cell lines as well as in 2 human cell lines and viral entry was blocked with sera from an HTLV-1 carrier and an adult T-cell leukemia patient. Unexpectedly, mouse cell lines EL4 and RLm1 and human cell lines Molt-4 and Hut78 showed similar efficiency for viral entry. These results suggest a wide distribution of HTLV-1 receptor in mouse cells.

Poly(ADP-ribose) polymerase (PARP) takes part mainly in regulation of DNA repair, thereby maintaining genomic stability in the nucleus. However, what role PARP plays in mitotic cells is not known. Centrosomes play an important role in maintaining the fidelity of chromosome distribution during cell division. Loss of these functions might cause chromosomal instability and aneuploidy. p53 and BRCA1 were recently found to localize to the centrosome at mitosis. We found that PARP is localized to the centrosomes and the chromosomes at cell-division phase and interphase by indirect immunofluorescence. Furthermore, by analysis of isolated centrosomes PARP protein was found to associate with the centrosomes during mitosis. These data suggest that PARP may be involved in maintenance of chromosomal stability.

Neuroblastoma is one of the most common malignant tumors of childhood and is characterized by regressive and progressive disease. Genetic factors that define progression of neuroblastomas are still unknown. We performed comparative genomic hybridization (CGH) on 27 neuroblastomas and dual-color fluorescence in situ hybridization (FISH) to identify genetic aberrations associated with progressive neuroblastoma showing resistance to aggressive treatment. 17q21-q25 gains and MYCN amplification were associated with stage 4 neuroblastomas; however, these genetic aberrations had no significant relation to the progression of stage 4 neuroblastomas. A novel chromosomal gain at 1q21-q25 was found in 8 of 16 cases (50%) of stage 4 neuroblastoma. Gain of 1q21-q25 was observed in all of the progressive cases (8/8), which showed resistance to chemotherapy, including 5 fatal neuroblastomas in stage 4, whereas 1q21-q25 gain was not found in any of the 8 remission cases in stage 4. Survival analysis also showed that 1q21-q25 gain was associated with a poor outcome. High xenotransplantability in nude mice was observed for the tumors with 1q21-q25 gain (4/5; 80%). These data show that 1q21-q25 gain is strongly associated with progression of stage 4 neuroblastoma. Furthermore, by dual-color FISH analysis using cosmid clones, the 1q21-q25 gain was narrowed to increase in DNA copy number on 1q23 in the fatal type of stage 4 neuroblastoma showing this gain. These results suggest that DNA amplification at 1q23 may play a role in the development of progressive neuroblastoma in an advanced stage.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that recognizes and binds to the nicks and ends of DNA, and catalyses successive ADP-ribosylation reactions. To clarify the function of PARP at the molecular level, we searched proteins which interact with PARP. In the auto-modification domain of PARP in Drosophila, there is a putative leucine-zipper motif which can interact with other protein molecules. To find interacting proteins we examined the auto-modification domain of Drosophila PARP, using the Far-Western screening method. From six independent cDNA clones isolated, we characterized two clones, PBP-3 and PBP-12. The predicted amino acid sequences from 109 to 269 of PBP-3 and from 184 to 312 of PBP-12 had more than 62% identities to mammalian L23a (rpl23a) and L22 (rpl22), the ribosomal proteins of the large subunit. This indicated that PBP-3 and PBP-12 are Drosophila homologues of L23a and L22, respectively. These Drosophila ribosomal protein L22 and L23a have additional Ala-, Lys- and Pro-rich sequences at the amino terminus, which have a resemblance to the carboxy-terminal portion of histone H1. Thus, Drosophila L22 and L23a might have two functions, namely the role of DNA-binding similar to histone H1 and the role of organizing the ribosome.

Analysis of human T-cell leukemia virus type 1 (HTLV-1)-infected cell types and the interplay of these infected cells in vivo should provide valuable information to elucidate the pathogenesis of HTLV-1-associated diseases in humans and in animal models. In this study, HTLV-1-infected cell types were identified in HTLV-1-infected C3H/HeJ mice. Pan T, CD 4 + , CD 8 + , granulocyte and pan B cell fractions in the splenocytes of MT-2 cell-inoculated mice were sorted by use of their cell surface high-density expression of CD CD 4 , CD 8 , Gr-1 and B 220 antigens, respectively, with a fluorescence-activated cell sorter. The pX sequence of HTLV-1 provirus in the lysate of each fraction was amplified by polymerase chain reaction and detected by Southern hybridization. Interestingly, in addition to the CD 4 + cell fraction, the pX sequence was also found in CD 8 + cell, B cell and granulocyte fractions. The hroad cell spectrum of HTLV-1 infection in mice is consistent with the situation in humans. Our findings indicate that HTLV-1 receptor or coreceptor is widely distributed among different cell types in mice.

Human T-cell leukemia virus type 1 (HTLV-1) is associated with adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and other diseases. For prevention of the transmission of HTLV-1 and manifestation of these diseases, a small-animal model, especially a mouse model, would be useful. We injected HTLV-1-producing T cells (MT-2) intraperitoneally into neonatal C3H/HeJ mice. While the antibody against HTLV-1 antigens was not detectable in C3H/HeJ mice, HTLV-1 provirus was frequently detected in the spleen, lymph nodes, and thymus by PCR. HTLV-1 provirus was present at the level of 0 to 30 molecules in 10 5 spleen cells at the age of 15 weeks. In addition, a 59-bp flanking sequence of the HTLV-1 integration site was amplified from the spleen DNA by linker-mediated PCR and was confirmed to be derived from the mouse genome. HTLV-1 provirus was found in the T-cell fraction of the mouse spleen. These results indicate that mice can be infected by HTLV-1 and could serve as an animal model for the study of HTLV-1 infection and its pathogenesis in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paraparesis. Inhibition of HTLV-1 transmission is important to prevent the above HTLV-1-associated diseases. We used the antisense oligodeoxynucleotides (oligos) complementary to the first splice junction, rex responsive site, gag, env, tax, rex, and p21 and evaluated the effects on the syncytium formation between HTLV-1 producing human T-cell line, C9/PL cells, and HTLV-1-uninfected human glioma cell line, U251-MG cells. The syncytium formation was significantly inhibited the virion production assayed by antisense oligos to env, tax, gag, p21, and rex, with antisense oligo to env being the most inhibitory. Antisense oligos to env and tax also inhibited reverse transcriptase activity. Antisense oligo to env may have a potential as a preventive measure of HTLV-1 replication and transmission in vivo.

Caspase activities and two cDNA sequences have been identified in Drosophila melanogaster. To study the molecular events following the activation of the apoptotic pathway in D. melanogaster, S2 cells were treated with etoposide and the timing of the apoptotic events, such as caspase activation, mitochondrial pore opening, and loss of membrane asymmetry, was determined. Poly(ADP-ribose) polymerase (PARP) is known to be cleaved in the early phase of apoptosis in vertebrate systems. Little is known about the involvement of PARP cleavage in apoptosis in invertebrates. If PARP inactivation is a general event, this could mean that DNA repair enzymes need to be cleaved for the death pathway to be completed. We have found that in etoposide-treated cells, PARP protein is processed, but the nature of the cleavage is not known. Further experiments must be conducted and the peptide fragments must be sequenced to relate protease activities with PARP cleavage.

Human T cell leukemia virus type 1 (HTLV-1) provirus integration was investigated in mice inoculated with MT-2 cells, a HTLV-1 producing human T-cell line. From spleen DNA derived from a MT-2 cell-injected mouse, we cloned a HTLV-1 integration site by ligation-mediated PCR. The nucleotide sequence showed that HTLV-1 provirus was integrated into an intron of the mouse transforming growth factor-α gene in the reverse orientation. This result provides for the first time molecular evidence that mice can be infected with HTLV-1.

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 u1. 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 glutathioneS-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 antiOpisthorchis viverrini antibody, smoking and alcohol drinking did not change the decreased risk. Other combinations of deoxyribonucleic acid-repair gene polymorphism and glutathione-Stransferases 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.

To define the involvement of p16/CDKN2 and p15/MTS2 inactivation in ovarian tumorigenesis and the association of these inactivation events with histological types and clinical stages of ovarian tumors, we analyzed homozygous deletion and somatic mutation of p16/CDKN2 and p15/MTS2 genes, as well as hypermethylation of the 5′-CpG island of the p16/CDKN2 gene, in 49 primary ovarian tumors and 6 ovarian carcinoma cell lines. We found homozygous deletions of p16/CDKN2 and p15/MTS2 in 6 (12%) and 5 (10%) primary tumors, respectively. Somatic mutation of p16/CDKN2 was found in only I primary tumor, but mutation of p15/MTS2 was not detected in any sample. None of the 28 primary tumors or 6 cell lines was hypermethylated at the 5′-CpG island of p16/CDKN2. The incidence of inactivation of p16/CDKN2 in primary tumors was significantly higher in the advanced stages (7 of 29) than in the early stages (0 of 14). Seven of 9 alterations in p16/CDKN2 and p15/MTS2 were observed in serous (3 of 12), endometrioid (3 of 9) and clear-cell (1 of 4) carcinomas. However, only normal sequences of these genes were detected in mucinous carcinomas. Loss of heterozygosity (LOH) at the IFNA locus was detected in 1 of 19 (5%) tumors, but no change at the D95171 locus was observed in 17 tumors. These results suggest that: (i) homozygous deletion is the main mechanism of inactivation of p16/CDKN2 and p15/MTS2 in ovarian tumorigenesis; (ii) inactivation of p16/CDKN2 and p15/MTS2 may be the histological type-specific events involved in ovarian tumorigenesis; and (iii) inactivation of p16/CDKN2 is potentially involved in the progression of ovarian tumors in advanced stages. © 1996 Wiley-Liss, Inc.

The heterotrimeric guanine-nucleotide-binding proteins (G proteins) and G protein-coupled hormone receptors including gonadotropin receptors have been suggested to play a role in ovarian tumorigenesis. However, no functional significance of gonadotropin receptors and G proteins in this process has been demonstrated. To investigate this issue, we examined point mutations in these genes in various types of ovarian tumors by polymerase chain reaction-single strand conformation polymorphism analysis and direct sequencing. Among 37 tumors (20 epitherial, 8 sex cord-stromal, and 9 germ cell tumors) and 5 carcinoma cell lines examined, no mutational sequence of G protein-interaction domains of luteinizing hormone receptor and follicle-stimulating hormone receptor, or "hot spots" of the alpha subunit of adenylyl cyclase-stimulating G protein and -inhibitory G protein was observed. Although this analysis was performed on only a limited number of tumors and cell lines, and on limited gene loci, the results suggest that mutational activation in gonadotropin receptors and G proteins is not crucial for ovarian tumorigenesis.

To analyze intrauterine transmission, MT-2 cells, a human T-cell line producing human T-cell leukemia virus type I (HTLV-I), were injected into eight pregnant F344 rats, and cesarean section was performed at day 23 of pregnancy. HTLV-I provirus was detected by PCR in the liver and spleen taken from one of the eight fetuses. Moreover, 71 offspring were delivered by cesarean section from the remaining seven dams and fostered by seven normal rats. HTLV-I provirus was detected in peripheral blood mononuclear cells in 2 of the 71 offspring 4 weeks after cesarean section. These results indicate for the first time the intrauterine transmission of HTLV-I. To confirm the postnatal transmission, MT-2 cells were injected into a dam within 24 h after delivery, and six offspring were fostered by this dam. HTLV-I provirus was detected in peripheral blood mononuclear cells of all six offspring. This animal model may be useful for analysis and prevention of mother-to-child transmission of HTLV-I.

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.

The early historical background of the discovery of poly(ADP-ribose) and the following development of science on poly(ADP- ribose) are reviewed. Fundamental knowledge on the natures of poly(ADP-ribose), poly(ADP-ribose) polymerase and enzymes degrading poly(ADP-ribose) are summarized with brief description on the methodology for their purification and characterization. Future prospect of research on biological significance of poly(ADP-ribose) has also been discussed briefly. (Mol Cell Biochem 138: 5–12, 1994)

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 G2/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. (Cancer Sci 2012; 103: 191–196)

The cell of origin of tumors and the factors determining the cell of origin remain unclear. In this study, a mouse model of precursor B acute lymphoblastic leukemia/lymphoma (pre-B ALL/LBL) was established by retroviral transduction of Myc genes (N-Myc or c-Myc) into mouse bone marrow cells. Hematopoietic stem cells (HSCs) exhibited the highest susceptibility to N-Myc-induced pre-B ALL/LBL versus lymphoid progenitors, myeloid progenitors and committed progenitor B cells. N-Myc was able to induce pre-B ALL/LBL directly from progenitor B cells in the absence of Ink4a and Arf. Arf was expressed higher in progenitor B cells than Ink4a. In addition, N-Myc induced pre-B ALL/LBL from Arf(-/-) progenitor B cells suggesting that Arf has a predominant role in determining the cell of origin of pre-B ALL/LBL. Tumor cells derived from Ink4a/Arf(-/-) progenitor B cells exhibited a higher rate of proliferation and were more chemoresistant than those derived from wild-type HSCs. Furthermore, the Mdm2 inhibitor Nutlin-3 restored p53 and induced massive apoptosis in mouse pre-B ALL/LBL cells derived from Ink4a/Arf(-/-) cells and human B-ALL cell lines lacking Ink4a and Arf expression, suggesting that Mdm2 inhibition may be a novel therapeutic approach to the treatment of Ink4a/Arf(-/-) B-ALL/LBL, such as is frequently found in Ph(+) ALL and relapsed ALL. Collectively, these findings indicate that Ink4a and Arf are critical determining factors of the cell of origin and the therapeutic sensitivity of Myc-induced lymphoid tumors.

Oxysterols are oxygenated derivatives of cholesterol generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by inflammation-associated non-enzymatic reactions. Oxysterol binding proteins (OSBPs) are cytosolic high affinity receptors for oxysterols. We previously found that OSBPL-8 is upregulated in liver fluke (Opisthorchis viverrini)-induced hamster cholangiocarcinoma (CCA). Our aims were to determine the expression patterns of OSBP isoforms in human CCA tissues and to evaluate whether OSBPs could be used as molecular markers for the identification of blood-borne CCA metastasis. Expression levels of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 in CCA tissues were detected using qRT-PCR and immunohistochemistry. Expression of OSBPs at mRNA level in the blood of CCA patients was also investigated. We confirmed increased expression of OSBPL-8 in O. viverrini -induced hamster CCA tissues. Moreover, increased expression of OSBP1, OSBP2, OSBPL-7 and OSBPL-8 was seen in human CCA tissues. Notably, a significant increased level of OSBPL-7 mRNA was observed in tumor compared to non-tumor liver tissue. Immunohistochemistry supported the mRNA results, in that OSBPL-7 protein was over-expressed in cancer cells and hepatocytes but not in normal biliary cells and surrounding inflammatory cells. Interestingly, in our preliminary results, significantly higher levels of OSBP2 and OSBPL-7 mRNA were seen in blood samples from CCA patients than in healthy controls. These results suggest that OSBP2 and OSBPL-7 might serve as molecular markers for the identification of CCA metastasis in the bloodstream.

PolyADP-ribosylation plays an essential function in maintenance of genomic stability and cell survival. Although there are several proteins served as acceptor proteins in vitro, there are few proteins in vivo that are identified, including poly(ADP-ribose) polymerase-1. We have been studying to analyze the mechanism of neuronal cell death observed in poly(ADP-ribose) glycohydrolase (PARG)-knockout Drosophila melanogaster that shows accumulation of polyADP-ribosylated proteins in the brain. As the first step, we have been trying to isolate the polyADP-ribosylated accepter proteins from the PARG-knockout fly. The strategy is to extract the polyADP-ribosylated proteins and isolate them with affinity chromatography using monoclonal antibody against poly(ADP-ribose) (PAR) (10H). The bound fraction was eluted by buffer containing salt. Next, part of eluted fraction is treated with NaOH for separating the proteins from PAR chain. Nontreated fraction and treated fraction were separated with two-dimensional gel electrophoresis. After protein staining, the specific spots that were newly found after NaOH treatment were candidate acceptor proteins for polyADP-ribosylation in vivo and could be analyzed with liquid chromatography-mass spectrometry. We present the procedure to this approach.

BACKGROUND Oral administration of low-dose etoposide is known to be effective against various malignancies, including malignant lymphoma. However, the effectiveness of low-dose etoposide as a treatment for adult T-cell leukemia (ATL) has not been established. METHODS A 74-year-old woman with ATL in acute phase was treated by daily oral administration of low-dose etoposide (25 mg/m2). The authors assayed changes in the surface markers and the amount of human T-cell lymphotropic virus type I (HTLV-I) proviral DNA in peripheral blood mononuclear cells (PBMC) by using flow cytometry and the polymerase chain reaction (PCR) method, respectively. RESULTS Before treatment, generalized lymphadenopathy and hepatomegaly were observed. In laboratory examination, the leukocyte count was 13.7 x 10(3)/microliters, with 65% abnormal lymphocytes. The percentages of CD3-, CD4-, and CD25-positive cells in PBMC were 84.4%, 84.4%, and 76.5%, respectively. The serum lactic dehydrogenase (LDH) level was 1376 IU/l (normal range, less than 520 IU/l). After the initiation of treatment, lymph-adenopathy and hepatomegaly disappeared, and the serum LDH level was reduced to the normal level before the 20th day of the treatment. On the 55th day of the treatment, CD25-positive cells had virtually disappeared. In addition, the amount of the proviral DNA in PBMC was reduced to approximately one-tenth by this treatment. Subsequently, the patient was in remission for more than 16 months. No side effects were observed. CONCLUSIONS Daily oral administration of low-dose etoposide can be a safe and effective treatment for patients with ATL. The authors believe this to be the first report of a patient with ATL in whom complete remission (CR) was achieved by this treatment.

The complete nucleotide (nt) sequence of the Xenopus laevis poly(ADP-ribose) polymerase (PARP)-encoding cDNA was determined. The putative X. laevis PARP protein consists of 1008 amino acids (aa) with a molecular weight of 113 kDa. X. laevis PARP shares 74, 83, 73, 78 and 42% aa sequence homology with the human, bovine, mouse, chicken and Drosophila melanogaster PARPs, respectively. Comparison of the PARP aa sequences among these species showed conservation of two zinc-finger motifs in the DNA-binding domain, and an NAD-binding motif and a Rossmann fold in the catalytic domain. The first Leu of the putative leucine zipper of D. melanogaster PARP is substituted to Lys in X. laevis PARP. All the Glu residues in the leucine zipper are conserved in these six species.

We have isolated and sequenced cDNAs encoding the catalytic domain of poly(ADP-ribose) polymerase (PARP) from Xenopus laevis and Oncorhyncus masou (cherry salmon). The cDNAs were amplified by polymerase chain reaction using heterologous oligonucleotides corresponding to the conserved sequences of mammalian cDNAs as primers. The deduced amino acid sequences of Xenopus laevis and cherry salmon cDNA showed 84.4% and 75.6% similarities to that of human PARP, respectively. In both species, mRNA for PARP was identified as a single band of 4 kb, and PARP mRNA was abundant in ovary and brain. Thus, mixed oligonucleotide-primed amplification is a useful method in the cloning of cDNAs from different species, and the catalytic domain of PARP is conserved structurally among phylogenetically different species, suggesting an importance of poly(ADP-ribosyl)ation.

Poly(ADP-ribose) glycohydrolase, extensively purified to homogeneity from nuclei of human placenta, is composed of a single polypeptide with a molecular mass of 71,000 daltons on sodium dodecyl sulfate-polyacrylamide gel. Judging from its physico-chemical and catalytic properties, the enzyme is similar to the nuclear glycohydrolase (glycohydrolase I), but not to the cytoplasmic glycohydrolase (glycohydrolase II) that has been purified from guinea pig liver (Tanuma, S., Kawashima, K., and Endo, H. (1986) J. Biol. Chem. 261, 965-969; Maruta, H., Inageda, K., Aoki, T., Nishina, H., and Tanuma, S. (1991) Biochemistry 30, 5907-5912). The rates of hydrolysis of (ADP-ribose)n bound to various proteins by the purified nuclear glycohydrolase were higher than those of the corresponding free polymers. Kinetic analyses revealed that the enzyme had more activity toward poly(ADP-ribose) bound to histone H1 or to poly(ADP-ribose) polymerase than toward oligo(ADP-ribose) bound to cytoplasmic proteins from mitochondria or mRNA ribonucleoprotein although the Km and Vmax values were dependent on the chain length (n). In contrast, cytoplasmic glycohydrolase purified from human erythrocytes was more active toward oligo(ADP-ribose) (n = 2.6 or 4.2) bound to the cytoplasmic proteins than to poly(ADP-ribose) (n = 14.6) bound to histone H1, and their kinetic parameters of glycohydrolase II were rather dependent on the acceptor molecules for (ADP-ribose)n. These results suggest that poly(ADP-ribose) glycohydrolase I may play an important role in regulation of poly(ADP-ribosyl)ation levels on chromosomal proteins in nuclei.

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/β-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.