Hyo Chol Ha

Poly(ADP-ribose) polymerase-1 dependence of stress-induced transcription factors and associated gene expression in glia

Poly(ADP-ribose) polymerase-1 (PARP-1, EC 2.4.2.30), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Excessive activation of PARP-1 by cellular insults depletes its substrate β-nicotinamide adenine dinucleotide and ATP, leading to cell death. PARP-1-deficient (PARP-1−/−) mice are protected from several forms of inflammation. In the present study, we demonstrate in PARP-1−/− glial cells a loss of several stress-activated transcription factors as well as decreased expression of genes for cytokines and cellular adhesion molecules. We also show that augmented expression of some of these genes is independent of PARP-1 catalytic activity. These findings indicate that PARP-1 plays a pivotal role in the initial inflammatory response by modulating transcription of inflammation-linked genes.

FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor β (TGF-β) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12−/−) mice manifest cell cycle arrest in G1 phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-β1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-β receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-β receptor construct. TGF-β receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12−/− cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12−/− cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-β receptor signaling.

Poly(ADP-ribose) polymerase-1 is required for efficient HIV-1 integration

Poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) is an abundant nuclear enzyme, activated by DNA strand breaks to attach up to 200 ADP-ribose groups to nuclear proteins. As retroviral infection requires integrase-catalyzed DNA strand breaks, we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP-1. Viral infection is almost totally abolished in PARP-1 knockout fibroblasts. This protection from infection reflects prevention of viral integration into the host genome. These findings suggest a potential for PARP inhibitors in therapy of HIV type I infection.

1-(N-Alkylamino)-11-(N-ethylamino)-4,8-diazaundecanes: Simple Synthetic Polyamine Analogues That Differentially Alter Tubulin Polymerization

Polyamine analogues such as bis(ethyl)norspermine and N1-(cyclopropylmethyl)-N11-ethyl-4,8-diazaundecane (CPENSpm) act as potent modulators of cellular polyamine metabolism in vitro and possess impressive antitumor activity against a number of cell lines. Some of these polyamine analogues appear to produce their cell-type-specific cytotoxic activity through the superinduction of spermidine/spermine N1-acetyltransferase (SSAT). However, there are several analogues (e.g., N1-(cycloheptylmethyl)-N11-ethyl-4, 8-diazaundecane (CHENSpm)) which are effective cytotoxic agents but do not superinduce SSAT. We have previously demonstrated that CPENSpm and CHENSpm both initiate the cell death program, although by different mechanisms, and that CHENSpm (but not CPENSpm) induces a G2/M cell cycle arrest. We now report that one potential mechanism by which some polyamine analogues can retard growth and ultimately produce cytotoxicity is through interference with normal tubulin polymerization. In these studies, we compare the effects of the polyamine analogues CHENSpm, CPENSpm, and (S)-N1-(2-methyl-1-butyl)-N11-ethyl-4,8-diazaundecane (IPENSpm) on in vitro tubulin polymerization. These spermine analogues behave very differently from spermine and from each other in terms of tubulin polymerization rate, equilibrium levels, and time of polymerization initiation. These results demonstrate that structurally similar polyamine analogues with potent antitumor effects can produce significantly different cellular effects. The discovery of polyamine analogues that can alter tubulin polymerization provides a series of promising lead compounds that may have a similar spectrum of activity to more difficult to synthesize compounds typified by paclitaxel.