Detection and quantification of poly-ADP-ribosylated cellular proteins of spleen and liver tissues of mice in vivo by slot and Western blot immunoprobing using polyclonal antibody against mouse ADP-ribose polymer

Poly-ADP-ribosylation (PAR) of cellular proteins has been shown to have decisive roles in diverse cellular functions including carcinogenesis. There are indications that metabolic level of poly-ADP-ribosylated cellular proteins might indicate carcinogenesis and, therefore, could be potentially used in cancer screening program. Keeping in mind the limitations of currently available assays of cellular PAR, a new assay is being reported that measures the metabolic level of poly-ADP-ribosylated cellular proteins. The ELISA based slot and Western blot immunoassay used polyclonal antibody against natural, heterogeneous ADP-ribose polymers. It could be successfully employed to qualitatively and quantitatively assay metabolic levels of poly-ADP-ribosylated proteins of spleen and liver tissues of normal mice or mice exposed to dimethylnitrosamine for up to 8 weeks; potentially PAR of cellular proteins could be assayed in any tissue or biopsy. Implications of the results in cancer screening program have been discussed. (Mol Cell Biochem 278: 213–221, 2005)     

State of the erythrocyte surface (echinocytosis) in experimental carcinogenesis]

Characteristic red cell deformation, echinocytosis peculiar to mammary tumour susceptible C3H mice, were revealed by a comparative study of erythrocytes in animals with a different natural resistance to spontaneous carcinogenesis. 58.6% of spiny red cells was found at the early latent period (at the age of 3-4 months) and reached 91.1% at the late latent period (at the age of 11-12 months). In the blood of intact C57BL/6 mice resistant to mammary carcinogenesis, at the same ages the echinocyte count ranged from 16.0 to 18.7%. The tumour growth (spontaneous tumour in C3H mice and transplantable Ehrlich adenocarcinoma in C57BL/6 mice was accompanied by an increase in the echynocyte count and in the expression of echinocytosis (up to 96.6 and 65.3%, respectively). Possible pathogenetic mechanisms of echinocytosis in carcinogenesis are discussed.     

The effect of exposure to carcinogenic metals on histone tail modifications and gene expression in human subjects

The precise mechanisms by which nickel and arsenic compounds exert their carcinogenic properties are not completely understood. In recent years, alterations of epigenetic mechanisms have been implicated in the carcinogenesis of compounds of these two metals. In vitro exposure to certain nickel or arsenic compounds induces changes in both DNA methylation patterns, as well as, in the levels of posttranslational modifications of histone tails. Changes in DNA methylation patterns have been reported in human subjects exposed to arsenic. Here we review our recent reports on the alterations in global levels of posttranslational histone modifications in peripheral blood mononuclear cells (PBMCs) of subjects with occupational exposure to nickel and subjects exposed to arsenic in their drinking water. Occupational exposure to nickel was associated with an increase in H3K4me3 and decrease in H3K9me2. A global increase in H3K9me2 and decrease in H3K9ac was found in subjects exposed to arsenic. Additionally, exposure to arsenic resulted in opposite changes in a number of histone modifications in males when compared with females in the arsenic population. The results of these two studies suggest that exposure to nickel or arsenic compounds, and possibly other carcinogenic metal compounds, can induce changes in global levels of posttranslational histone modifications in peripheral blood mononuclear cells.     

An enzyme-linked immunosorbent poly(ADP-ribose) polymerase biomarker assay for clinical trials of PARP inhibitors

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. The DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is activated by DNA damage and has critical roles in DNA repair. Inhibition of PARP potentiates the activity of DNA-damaging agents such as temozolomide, topoisomerase inhibitors and radiation in both in vitro and in vivo preclinical models. Recently, several PARP inhibitors have entered clinical trials either as single agents or in combination with DNA-damaging chemotherapy. Because PARP inhibitors are not cytotoxic, a biomarker assay is useful to guide the selection of an optimal biological dose. We set out to develop an assay that enables us to detect 50% PAR reduction in human tumors with 80% power in a single-plate assay while assuring no more than a 10% false-positive rate. We have developed and optimized an enzyme-linked immunosorbent assay (ELISA) to measure PARP activity that meets the above-mentioned criterion. This robust assay is able to detect PAR levels of 30-2000 pg/ml in both tumor and peripheral blood monocyte samples. In a B16F10 mouse syngeneic tumor model, PARP inhibitor ABT-888 potentiates the effect of temozolomide in suppressing tumor growth, and PARP activity is greatly reduced by ABT-888 at efficacious doses. In summary, the ELISA assay described here is suitable for biomarker studies in clinical trials of PARP inhibitors.     

ADP-Ribosylation: Activation,Recognition, and Removal

ADP-ribosylation is a type of posttranslational modification catalyzed by members of the poly(ADP-ribose) (PAR) polymerase superfamily. ADP-ribosylation is initiated by PARPs, recognized by PAR binding proteins, and removed by PARG and other ADP-ribose hydrolases. These three groups of proteins work together to regulate the cellular and molecular response of PAR signaling, which is critical for a wide range of cellular and physiological functions.     

Influence of chain length and branching on poly(ADP-ribose)–protein interactions

Hundreds of proteins interact with poly(ADP-ribose) (PAR) via multiple PAR interaction motifs, thereby regulating their physico-chemical properties, sub-cellular localizations, enzymatic activities, or protein stability. Here, we present a targeted approach based on fluorescence correlation spectroscopy (FCS) to characterize potential structure-specific interactions of PAR molecules of defined chain length and branching with three prime PAR-binding proteins, the tumor suppressor protein p53, histone H1, and the histone chaperone APLF. Our study reveals complex and structure-specific PAR–protein interactions. Quantitative K_d values were determined and binding affinities for all three proteins were shown to be in the nanomolar range. We report PAR chain length dependent binding of p53 and H1, yet chain length independent binding of APLF. For all three PAR binders, we found a preference for linear over hyperbranched PAR. Importantly, protein- and PAR-structure-specific binding modes were revealed. Thus, while the H1-PAR interaction occurred largely on a bi-molecular 1:1 basis, p53—and potentially also APLF—can form complex multivalent PAR–protein structures. In conclusion, our study gives detailed and quantitative insight into PAR–protein interactions in a solution-based setting at near physiological buffer conditions. The results support the notion of protein and PAR-structure-specific binding modes that have evolved to fit the purpose of the respective biochemical functions and biological contexts.     

Poly(ADP-ribosyl)ation-dependent Transient Chromatin Decondensation and Histone Displacement following Laser Microirradiation

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.     

Role of mouse spleen cell HMG proteins and their poly-ADP-ribosylation in betelnut induced carcinogenesis

The role of high mobility group (HMG) proteins and their poly-ADP-ribosylation (PAR) in betel nut induced initiation of carcinogenesis in mice has been studied. A known carcinogen, diethylnitrosamine (DEN) was used as a positive control. Swiss albino mice were chronically exposed to aqueous extract of betel nut (AEBN) or DEN at low doses for up to 4 weeks. The poly-ADP-ribosylation (PAR) of spleen cell HMG proteins was monitored using [32P]-NAD+. Parallel to this, chromatin was subjected to DNase I cleavage and the organizational state of the chromatin was monitored. The PAR of HMG proteins showed a marked progressive reduction at different times following AEBN- or DEN treatment. HMG proteins isolated from the control and carcinogen treated mice were allowed to reassociate with the untreated spleen cells chromatin. The reassociated chromatin showed progressive relaxation in its superstructure. The results suggest that under the influence of potential carcinogens AEBN or DEN, the mouse spleen cell HMG proteins created molecular conditions favourable to initiation of cancer.     

Transformation of NIH 3T3 cells by enhanced PAR expression

Prostate androgen regulated (PAR) is a 1038bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.     

Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation.Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment.PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome.Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as providing the basis for selection of the appropriate genetic model organisms to study the physiology of the specific human PARP proteins.     

Poly(ADP-ribose) metabolism is essential for proper nucleoprotein exchange during mouse spermiogenesis

Sperm chromatin is organized in a protamine-based, highly condensed form, which protects the paternal chromosome complement in transit, facilitates fertilization, and supports correct gene expression in the early embryo. Very few histones remain selectively associated with genes and defined regulatory sequences essential to embryonic development, while most of the genome becomes bound to protamine during spermiogenesis. Chromatin remodeling processes resulting in the dramatically different nuclear structure of sperm are poorly understood. This study shows that perturbation of poly(ADP-ribose) (PAR) metabolism, which is mediated by PAR polymerases and PAR glycohydrolase in response to naturally occurring endogenous DNA strand breaks during spermatogenesis, results in the abnormal retention of core histones and histone linker HIST1H1T (H1t) and H1-like linker protein HILS1 in mature sperm. Moreover, genetic or pharmacological alteration of PAR metabolism caused poor sperm chromatin quality and an abnormal nuclear structure in mice, thus reducing male fertility.     

Fractionated low-dose radiation exposure leads to accumulation of DNA damage and profound alterations in DNA and histone methylation in the murine thymus

Thymus, an important component of hematopoietic tissue, is a well-documented "target" of radiation carcinogenesis. Both acute and fractionated irradiation result in a high risk of leukemia and thymic lymphoma. However, the exact mechanisms underlying radiation-induced predisposition to leukemia and lymphoma are still unknown, and the contributions of genetic and epigenetic mechanisms in particular have yet to be defined. Global DNA hypomethylation is a well-known characteristic of cancer cells. Recent studies have also shown that tumor cells undergo prominent changes in histone methylation, particularly a substantial loss of trimethylation of histone H4-Lys20 and demethylation of genomic DNA. These losses are considered a universal marker of malignant transformation. In the present study, we investigated the effect of low-dose radiation exposure on the accumulation of DNA lesions and alterations of DNA methylation and histone H4-Lys20 trimethylation in the thymus tissue using an in vivo murine model. For the first time, we show that fractionated whole-body application of 0.5 Gy X-ray leads to decrease in histone H4-Lys20 trimethylation in the thymus. The loss of histone H4-Lys20 trimethylation was accompanied by a significant decrease in global DNA methylation as well as the accumulation of DNA damage as monitored by persistence of histone gammaH2AX foci in the thymus tissue of mice exposed to fractionated irradiation. Altered DNA methylation was associated with reduced expression of maintenance (DNMT1) and, to a lesser extent, de novo DNA methyltransferase DNMT3a in exposed animals. Expression of another de novo DNA methyltransferase DNMT3b was decreased only in males. Irradiation also resulted in approximately 20% reduction in the levels of methyl-binding proteins MeCP2 and MBD2. Our results show the involvement of epigenetic alterations in radiation-induced responses in vivo. These changes may play a role in genome destabilization that ultimately leads to cancer.     

Polarity-regulating kinase partitioning-defective 1b (PAR1b) phosphorylates guanine nucleotide exchange factor H1 (GEF-H1) to regulate RhoA-dependent actin cytoskeletal reorganization

Partitioning-defective 1b (PAR1b), also known as microtubule affinity-regulating kinase 2 (MARK2), is a member of evolutionally conserved PAR1/MARK serine/threonine kinase family, which plays a key role in the establishment and maintenance of cell polarity at least partly by phosphorylating microtubule-associated proteins (MAPs) that regulate microtubule stability. PAR1b has also been reported to influence actin cytoskeletal organization, raising the possibility that PAR1b functionally interacts with the Rho family of small GTPases, central regulators of the actin cytoskeletal system. Consistent with this notion, PAR1 was recently found to be physically associated with a RhoA-specific guanine nucleotide exchange factor H1 (GEF-H1). This observation suggests a functional link between PAR1b and GEF-H1. Here we show that PAR1b induces phosphorylation of GEF-H1 on serine 885 and serine 959. We also show that PAR1b-induced serine 885/serine 959 phosphorylation inhibits RhoA-specific GEF activity of GEF-H1. As a consequence, GEF-H1 phosphorylated on both of the serine residues loses the ability to stimulate RhoA and thereby fails to induce RhoA-dependent stress fiber formation. These findings indicate that PAR1b not only regulates microtubule stability through phosphorylation of MAPs but also influences actin stress fiber formation by inducing GEF-H1 phosphorylation. The dual function of PAR1b in the microtubule-based cytoskeletal system and the actin-based cytoskeletal system in the coordinated regulation of cell polarity, cell morphology, and cell movement.     

Comparing the response of Antarctic,tropical and temperate microalgae to ultraviolet radiation (UVR) stress

The response of Antarctic, tropical and temperate microalgae of similar taxonomic grouping to ultraviolet radiation (UVR) stress was compared based on their growth and fatty acid profiles. Microalgae of similar taxa from the Antarctic (Chlamydomonas UMACC 229, Chlorella UMACC 237 and Navicula UMACC 231), tropical (Chlamydomonas augustae UMACC 246, Chlorella vulgaris UMACC 001 and Amphiprora UMACC 259) and temperate (Chlamydomonas augustae UMACC 247, Chlorella vulgaris UMACC 248 and Navicula incerta UMACC 249) regions were exposed to different UVR conditions. The cultures were exposed to the following conditions: PAR (42 μmol photons m⁻² s⁻¹), PAR + UVA (854 μW cm⁻²) and PAR + UVA + UVB (117 μW cm⁻²). The cultures were subjected to UVA doses of 46.1, 92.2 and 184.4 J cm⁻² and UVB doses of 6.3, 12.6 and 25.2 J cm⁻² by varying the duration of their exposure (1.5, 3 and 6 h) to UVR during the light period (12:12 h light-dark cycle). UVA did not affect the growth of the microalgae, even at the highest dose. In contrast, growth was adversely affected by UVB, especially at the highest dose. The dose that caused 50% inhibition (ID₅₀) in growth was used to assess the sensitivity of the microalgae to UVB. Sensitivity of the microalgae to UVB was species-dependent and also dependent on their biogeographic origin. Of the nine microalgae, the Antarctic Chlorella was most tolerant to UVB stress (ID₅₀ = 21.0 J cm⁻²). Except for this Chlorella, the percentage of polyunsaturated fatty acids of the microalgae decreased in response to high doses of UVB. Fatty acid profile is a useful biomarker for UVB stress for some microalgae. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of mice

The incorporation of the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) into hair of newborn mice was investigated in order to contribute to the validation of PhIP in hair as a suitable biomarker for human dietary exposure. Black mice (C57BL/6J; 7-9 days old) were given graded doses of [³H]-PhIP subcutaneously during the start of the hair growth period. The distribution of [³H]-PhIP and incorporation into hair were investigated by tape-section autoradiography. Almost all the radioactivity in hair represented PhIP as shown by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). A dose-response proportionality of incorporation into hair was found when incorporation was determined by liquid scintillation counting. Autoradiography showed that PhIP was rapidly cleared from the skin, but remained for at least 28 days in the part of the hair shafts which was formed during the exposure period. The present results obtained using the mouse as a model, further support the suggestion that PhIP in hair may be a suitable biomarker for human exposure to dietary PhIP.     

Immunogenetic differences between lymph node cell and bone marrow cell grafts in irradiated mice

C3H lymph node cell (LNC) grafts, but not bone marrow cell (BMC) grafts, were resisted by lethally irradiated NZB, (C57BL × NZB)F1, and (C57BL/6 × DBA/2)F1 mice. BALB/ c hosts did not resist C3H LNC, suggesting that Ir-like genes regulate resistance to such grafts. Cyclophosphamide, silica particles, and ⁸⁹Sr pretreatments of prospective host mice resulted in successful proliferation of C3H LNC in most instances. These agents were known to abrogate resistance to incompatible BMC grafts. The determinants for antigens recognized on LNC appear to map in or near the D region of H-2. LNC grafts of all H-2^k strains tested (C3H, CBA, C58, C57BR) were strongly resisted while A, C3H.A, B10.A(5R), A.TL, and A.Tla^b LNC grafts were not strongly resisted by NZB hosts. Grafts of H-2^b (C57BL/6, C57BL/10, 129) LNC, or BMC are resisted by NZB or (C57BL/6 × DBA/2)F1 hosts. (C3H × C57BL)F1 LNC but not BMC were resisted by similar hosts. (C57BL/6 × DBA/2)F1 mice were injected with C57BL/6 spleen cells four times to induce specific “unresponsiveness” to parental-strain Hemopoietic histocompatibility (Hh) antigens. Unresponsiveness was induced to C57BL/6 BMC, as expected, but C57BL/6 and C3H LNC grafts were resisted despite the spleen cell injections. The data suggest that the antigens recognized during rejection of C3H LNC are not expressed on C3H BMC. It is even conceivable that Hh antigens on C57BL/6 BMC and LNC have separate determinants. Alternatively, the injections of C57BL/6 spleen cells may have induced an anti-idiotypic response that was capable of eliminating C57BL/6 LNC by a different effector mechanism.     

Loss of transgelin in repeated bouts of ulcerative colitis-induced colon carcinogenesis

Though ulcerative colitis (UC)-associated colon cancer develops from dysplastic lesions caused by chronic inflammation, the specific mechanistic link between chronic inflammation and carcinogenesis in colon has not been integrated into molecular understanding. We therefore established an experimental animal model for colitic cancer, and used proteomic analysis, based on 2-DE and MALDI-TOF MS, to identify proteins involved in colitic cancer. In our model, 6-week-old C57BL/6J mice were exposed to 15 cycles of dextran sodium sulfate (DSS), with each cycle consisting of 0.7% DSS for 1 week followed by distilled water for 10 days. Colorectal tumors developed in 22 of 24 mice (91.6%), with a tumor multiplicity of 1.727 per tumor-bearing mouse. Comparative 2-DE analysis showed that 38 protein spots were differentially expressed in colon tumors and normal colon. We identified 27 of these proteins, including GRP94, HSC70, enolase, prohibitin, and transgelin. The reduction of transgelin expression in mouse colon tumors was confirmed by Western blotting and immunohistochemistry. We also found that transgelin expression was significantly reduced in human colon tumors compared with adjacent nontumorous tissues. In conclusion, these results suggest that loss of transgelin could be a candidate for biomarker of repeated colitis-associated colon cancer.