Piera Quesada

Poly(ADPR) polymerase-1 and poly(ADPR) glycohydrolase level and distribution in differentiating rat germinal cells

Poly(ADP-ribose)polymerase (PARP-1) and poly(ADP-ribose)glycohydrolase (PARG) are responsible for the transient poly(ADP-ribosyl)ation of proteins in eukaryotic cells. This biochemical reaction plays an active role in DNA replication and repair, transcription, cell differentiation and death. The aim of this study was to investigate the levels and the sub-cellular distribution of such enzymes in rat germinal cells at different stages of differentiation, i.e. in primary spermatocytes and round spermatids, representing meiotic and post-meiotic cells, respectively. The determination of the level of PARP-1 mRNA and protein revealed its higher expression in primary spermatocytes, thus implying that PARP-1 is one of the meiotic genes whose expression is requested at the pachytene phase of the meiosis. We also demonstrated that rat germinal cells contain both the forms of PARG (i.e. of 110 and 60 kDa) so far described in somatic cells. In our experimental system, the large PARG was present and active mainly in the nuclear fraction of primary spermatocytes, whereas round spermatids showed a higher level of the 60 kDa PARG in the post-nuclear fraction. Collectively, our data show a different expression level of PARP-1 and a different endocellular distribution of PARG and suggest a role for the poly(ADP-ribose) turnover in distinct pathways in meiotic and post-meiotic germinal cells.

Poly(ADP-ribose) binding properties of histone H1 variants

Using a poly(ADP-ribose) binding assay on protein blots we examined the ability of rat testis histone H1 variants to establish non-covalent interactions with the polymer. All the H1 variants bound ADP-ribose polymers; the binding was salt resistant and highly specific, occurring even in the presence of a large excess of competitor DNA. A comparison among the H1 variants showed that H1t has the highest affinity for poly(ADP-ribose). Long and branched poly(ADP-ribose) molecules were found to be preferentially involved in the interaction with the histone variants. The results further corroborate the concept that non-covalent interactions of poly(ADP-ribose) with target proteins may constitute an important mechanism to modulate chromatin structure.

Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment.

The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids. Using a comet assay at pH 10.0 to assess single strand breakage (SSB) in DNA, it was found that a high level of damage was induced by 5 Gy gamma-irradiation and acute exposure to 50 microM H2O2. This damage was effectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(ADP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA). Immunofluorescence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids. Poly(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma-irradiation or H2O2 treatment. Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stresses. The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathione (SNOG), caused significant SSBs in both spermatocytes and spermatids. The effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions. SNOG-induced SSBs were insensitive to both CAT and SOD. It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma-irradiation and H2O2 and that efficient repair of SSBs requires PARP activity.

Poly(ADP-ribosylation) of nuclear proteins in rat testis correlates with active spermatogenesis

Poly(ADP-ribosylation) of nuclear proteins has been investigated in rat testis under different experimental conditions to determine whether it is associated with somatic or germinal cells. Isolated, intact nuclei were incubated with [14C]NAD and extracted sequentially with 5% HClO4 and 0.25 M HCl, and labelled soluble proteins were analysed by reverse-phase high-performance liquid chromatography and acetic acid-urea polyacrylamide gel electrophoresis (pH 2.9). Results show that in normal adult testis a major acceptor protein for poly(ADP-ribose) in HClO4 extracts is the tissue-specific histone, H1t. Core histones and three proteins (alpha, beta and gamma) with low mobility on acetic acid-urea gels were the major acceptors identified in HCl extracts. Poly(ADP-ribosylation) of all the aforementioned proteins is very low in isolated intact nuclei of testis from 8-day-old animals (only spermatogonia present in seminiferous tubules), increases significantly by 16-day (pachytene spermatocytes appear) and reaches adult proportions by 32 days (condensing spermatids present). In the nuclei from cryptorchid testes, poly(ADP-ribosylation) of nuclear proteins resembles 8-day-old testis. It is concluded that (a) poly(ADP-ribosylation) of nuclear proteins in rat testis is closely correlated with spermatogenesis and can be inferred that is particularly active in the early stages of meiosis; (b) testis-specific proteins (histone H1t and low mobility proteins, alpha, beta and gamma) are poly(ADP-ribosylated) to higher specific radioactivity than somatic histones.

Co-localization of poly(ADPR)polymerase 1 (PARP-1) poly(ADPR)polymerase 2 (PARP-2) and related proteins in rat testis nuclear matrix defined by chemical cross-linking

Poly(ADPR)polymerase 1 and 2 (PARP‐1, PARP‐2) are nuclear enzymes which function is based on specific interactions with DNA and nuclear proteins. PARPs targets include proteins involved in DNA replication, repair, and transcription and their function can be modulated either by protein–protein interaction with native PARP‐1 and by non‐covalent interaction with poly(ADP‐ribose) (pADPR) linked to the auto‐modified PARP‐1. Moreover, the association of pADPR and PARP‐1 with the nuclear matrix (NM) has been reported, based on the poly(ADP‐ribosyl)ation of nuclear matrix proteins (NMPs). In the present article, by the use of DNA and protein cross‐linking reactions, by cis‐diamminedichloroplatinum II (cDDP) and sodium tetrathionate (NaTT) respectively, we present more evidences about the association of PARP‐1, PARP‐2, and PARPs related proteins with the NM. Our findings confirmed that NM could be seen as a fraction greatly enriched in transcription factors (i.e., C/EBP‐β) and enzymes (DNA Topo II, DNA PK) that co‐localize with PARP‐1 and ‐2 at the matrix associated regions (MARs) of chromatin. Moreover, pADPR contributes to PARP‐1 localization at the NM, showing that PARP(s) activity co‐operates to the functions of this nuclear fraction.

The analysis of the poly(ADPR) polymerase mode of action in rat testis nuclear fractions defines a specific poly(ADP-ribosyl)ation system associated with the nuclear matrix.

The poly(ADP-ribosyl)ation system, associated with different nuclear fractions of rat testis, has been analyzed for both pADPR and pADPR acceptor proteins. The DNase I sensitive and resistant chromatin contain 35% and 40%, respectively, of the total pADPR synthesized in intact nuclei incubated with [32P]NAD. Moreover, the residual 25% were estimated to be associated with the nuclear matrix.Three different classes of pADPR are present in the nuclei. The longest and branched ADPribose polymers modify proteins present in the DNase I resistant (2 M NaCl extractable) chromatin and in the nuclear matrix, whereas polymers of > 20 residues interact with the components of the DNase I sensitive chromatin and oligomers of 6 ADPribose residues are bound specifically to the acid-soluble chromosomal proteins, present in isolated nuclear matrix. The main pADPR acceptor protein in all the nuclear fractions is represented by the PARP itself (auto-modification reaction). The hetero-modification reaction occurs mostly on histone H1 and core histones, that have been found associated to DNase I sensitive and resistant chromatin, respectively. Moreover, an oligo(ADP-ribosyl)ation occurs on core histones tightly-bound to the matrix associated regions (MARs) of chromatin loops.

Poly(ADP-ribose) polymerase signaling of topoisomerase 1-dependent DNA damage in carcinoma cells

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOP1) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity. In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2(+/+) and p53(+/+). HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TPT+PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT+PJ34 combined treatment; (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24-72h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors.

ADP-ribosylation of nuclear proteins in rat ventral prostate during ageing

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.

ADP-ribosylation reactions in Sulfolobus solfataricus, a thermoacidophilic archaeon

An ADP-ribosylating system was detected in a crude homogenate from Sulfolobus solfataricus, a thermophilic archaeon, optimally growing at 87 degrees C. The archaeal ADP-ribosylation reaction was time-, temperature- and NAD-dependent. It proved to be highly thermostable, with about 30% decrease of 14C incorporation from [14C]NAD on incubation at 80 degrees C for up to 24 h. The main reaction product was found to be mono-ADP-ribose. Testing both [adenine-14C(U)]NAD and [adenine-14C(U)]ADPR as substrates, it was found that acceptor proteins were modified by ADP-ribose both enzymatically, via ADP-ribosylating enzymes, and via chemical attachment of free ADP-ribose, likely produced by NAD glycohydrolase activity. The synthesis of ADP-ribose-protein complexes was shown to involve mainly acceptors with molecular masses in the 40-100 kDa range, as determined by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulphate.

Poly(ADP‐ribose) polymerase‐1: Association with nuclear lamins in rodent liver cells

The distribution of poly(ADP‐ribose) polymerase‐1 (PARP‐1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A‐ and B‐type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix‐associated PARP‐1 partitioned, in an active form, with the insoluble, lamin‐enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP‐1–protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP‐1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X‐100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co‐immunoprecipitation experiments our results show that the lamins are prominent PARP‐1‐binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix.