Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.

The antitumor antibiotic and cancer chemotherapeutic agent mitomycin C (MC) alkylates and crosslinks DNA, forming six major MC-deoxyguanosine adducts of known structures in vitro and in vivo. Two of these adducts are derived from 2,7-diaminomitosene (2,7-DAM), a non-toxic reductive metabolite of MC formed in cells in situ. Several methods have been used for analysis of MC-DNA adducts in the past; however, a need exists for a safer, more comprehensive and direct assay of the six-adduct complex. Development of an assay, based on mass spectrometry is described. DNA from EMT6 mouse mammary tumor cells, Fanconi Anemia -A fibroblasts, normal human fibroblasts, and MCF-7 human breast cancer cells was isolated after MC or DMC treatment of the cells, digested to nucleosides and submitted to liquid chromatography electrospray-tandem mass spectrometry. Two fragments of each parent ion were monitored ("multiple reaction monitoring"; MRM). Identification and quantitative analysis was based on a standard mixture of six adducts, the preparation of which is described here in detail. The lower limit of detection of adducts is estimated as 0.25 picomol. Three initial applications of this method are reported: (i) differential kinetics of adduct repair in EMT6 cells; (ii) analysis of adducts in MC-or DMC-treated Fanconi Anemia cells; and (iii) comparison of the adducts generated by treatment of MCF-7 breast cancer cells with MC and DMC. Notable results are the following: repair removal of the DNA interstrand cross-link and of the two adducts of 2,7-DAM is relatively slow; both MC and DMC generate DNA interstrand cross-links in human fibroblasts, Fanconi Anemia-A fibroblasts and MCF-7 cells as well as EMT6 cells; DMC shows a stereochemical preference of linkage to the guanine-2-amino group opposite from that of MC. This information is available free of charge via the Internet at

10-Decarbamoyl-mitomycin C (DMC), a mitomycin C (MC) derivative, generates an array of DNA monoadducts and interstrand cross-links stereoisomeric to those that are generated by MC. DMC was previously shown in our laboratory to exceed the cytotoxicity of MC in a human leukemia cell line that lacks a functional p53 pathway (K562). However, the molecular signal transduction pathway activated by DMCDNA adducts has not been investigated. In this study, we have compared molecular targets associated with signaling pathways activated by DMC and MC in several human cancer cell lines. In cell lines lacking wild-type p53, DMC was reproducibly more cytotoxic than MC, but it generated barely detectable signal transduction markers associated with apoptotic death. Strikingly, DMCs increased cytotoxicity was not associated with an increase in DNA double-strand breaks but was associated with early poly(ADP-ribose) polymerase (PARP) activation and Chk1 kinase depletion. Alkylating agents can induce increased PARP activity associated with programmed necrosis, and the biological activity of DMC in p53-null cell lines fits this paradigm. In cell lines with a functional p53 pathway, both MC and DMC induced apoptosis. In the presence of p53, both MC and DMC activate procaspases; however, the spectrum of procaspases involved differs for the two drugs, as does induction of p73. These studies suggest that in the absence of p53, signaling to molecular targets in cell death can shift in response to different DNA adduct structures to induce non-apoptotic cell death.

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-β-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-β-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-β stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.

Regulation of NF-kB nuclear translocation and stability is central to mounting an effective innate immune response. Here, we describe a novel molecular mechanism controlling NF-kB-dependent innate immune response. We show that a previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila. Charon is an ankyrin repeat-containing protein that mediates PARP-1-dependent transcriptional responses downstream of the innate immune pathway. Our results demonstrate that Charon interacts with the NF-kB orthologue Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-kB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, our results are the first to describe the molecular mechanism regulating translocation of the NF-kB subunit from cytoplasm to chromatin.

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This study shows that melanogenesis can be induced by AGI-1140 without many of the deleterious effects associated with UVB.

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