Mark E. Smulson

Regulation of α-synuclein expression by poly (ADP ribose) polymerase-1 (PARP-1) binding to the NACP-Rep1 polymorphic site upstream of the SNCA gene

Alleles at NACP-Rep1, the polymorphic microsatellite repeat located approximately 10 kb upstream of the alpha -synuclein gene (SNCA), are associated, in some reports, with differing risks of sporadic Parkinson disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of SNCA have been implicated in familial Parkinson disease (PD), even a 1.5-2-fold increase in alpha -synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1s catalytic domain increased the endogenous SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1s catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on SNCA expression.

DNA transitions induced by binding of PARP‐1 to cruciform structures in supercoiled plasmids

Poly(ADP‐ribose)polymerase‐1 (PARP‐1) binds to single and double‐stranded breaks in DNA, but less well known is its affinity for undamaged DNA. Previously, we have shown that PARP‐1 also binds to the hairpin structures in DNA models. The mechanism underlying these interactions remains to be defined.

Phenoxodiol inhibits growth of metastatic prostate cancer cells

Phenoxodiol, a synthetic analog of Genistein, is being assessed in several clinical studies against a range of cancer types and was shown to have a good efficacy and safety profile. In this study we tested the effects of Phenoxodiol against prostate cancer cell lines.

A dot-blot method for screening polyclonal and monoclonal antisera to poly(ADP-ribose)

ADP-ribosylation reactions play a key role at several points in cellular regulation and repair of DNA damage. The use of polyclonal or monoclonal antisera to poly(ADP-ribose) as probes to localize the site(s) of action of the polymer offers a promising tool for these studies. We report here a simple, sensitive method for detection and titration of these antisera to poly(ADP-ribose) using nitrocellulose membrane (NC) as a support for a dot-blot analysis. We take advantage of the fact that a highly labeled poly(ADP-ribose) preparation can be obtained by incubation of a 0.3 M KCl extract prepared from calf thymus nuclei with 32P-NAD. Such a preparation of labeled antigen is used as a reagent to detect the positive antibody spots on the NC with negligible background. Subsequent titration of the antisera and their semi-quantitative evaluation are also feasible using the dot-blot method. The sensitivity of the assay is only limited by the specific activity that can be achieved for the labeled polymer prepared as the antigen probe. The advantage of this method is that it eliminates the need to prepare pure, highly radiolabeled polymer as well as the fact that several samples can be handled on the membrane simultaneously. We demonstrate application of this technique for screening sera from patients with systemic lupus erythematosus (SLE) for anti-poly(ADP-ribose) antibodies. Further, we also extend the use of these sera for immunoquantitation of ADP-ribosylated proteins in six human tumor cells in tissue culture.

Detection of heterogeneity of apoptotic fragments of poly (ADP‐ribose) polymerase in MDA‐MB‐468 breast cancer cells: Two‐dimensional gel analysis

Caspace‐mediated proteolysis of the nuclear enzyme poly(ADP‐ribose) polymerase (PARP) (EC 2.4, 2.30) is a biochemical marker of cell death in response to various apoptotic stimuli. Anti‐PARP antibodies identifying the 89 kDa polypeptide from the C‐terminus as well as the 113 kDa native enzyme are often used to demonstrate evidence of apoptosis‐associated, interleukin converting enzyme (ICE)‐mediated limited cleavage. Recent evidence points to redundancy of caspases, heterogeneity of their cleavage sites, and a possibility of generating distinct context‐specific, and cell‐specific PARP fragments. In the present study, we employed antibodies directed to multiple sites in PARP and probed two‐dimensionally resolved proteins of the estrogen receptor negative MDA‐MB‐468 breast tumor cells, induced to undergo apoptosis by ionizing radiation (IR). Our results revealed that the 24 kDa apoptotic fragment of PARP, from the N‐terminus, consists of at least three isoforms, located at a pI more basic than the full length enzyme. We also report a hitherto unrecognized feature of an anti‐PARP antiserum, VIC‐5, detecting both the 89 kDa and the 24 kDa caspase‐generated fragments of PARP. Thus, application of two‐dimensional electrophoresis combined with antisera directed to multiple sites would be valuable in distinguishing PARP cleavage site‐ and inhibitor specificities of proteases during apoptosis.

The association of human c-Ha-ras sequences with chromatin and nuclear proteins

As a step towards the understanding of possible relationship between chromatin organization and regulation of the oncogene expression, we have investigated the chromatin structure of one of the more frequently activated oncogenes, c-Ha-ras, in HeLa-S3 cells. This was accomplished by isolation of the chromatin fractions (soluble and insoluble) after micrococcal nuclease digestion of purified nuclei and probing for the distribution of ras sequences. The polynucleosomal fraction was further resolved by sucrose gradient sedimentation. Southern-blot hybridization of the DNA isolated from various fractions yielded following results: (1) c-Ha-ras sequences segregated predominantly in the lysate fraction. (2) Unlike the B-globin (transcriptionally inactive) sequences, ras-H associated chromatin lacked typical nucleosomal packaging. Furthermore, since post-translational modifications of nuclear proteins have been suggested to modulate the nucleosome structure during DNA transcription and replication, ras sequences, in polynucleosomes immunofractionated on anti-poly (ADP-Ribose) Sepharose were also examined. The data suggested that the major class of this oncogene sequence exists in chromatin more distal to the sites of this particular chromatin modification.

Radiation Sensitivity of Human Cells and the Poly(ADP-Ribose) Polymerase Gene

Until recently much of radiation biology has centered about cellular responses to radiation using the clonogenic assay to study radiation sensitivity and radiation repair processes. The availability of molecular techniques, allowing direct genomic analysis of cellular function, permits a much more detailed view of events taking place in the DNA as a result of radiation exposure. The development of a complementary cDNA to the poly(ADP-ribose) polymerase gene (3) has provided a unique opportunity to study the genomic events associated with poly(ADP-ribose) metabolism.