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Several 2',3'-dideoxy-3'-thiapyrimidine nucleosides were studied for their ability to inhibit hepatitis B virus (HBV) DNA replication in a HBV-transfected cell line (2.2.15). 2',3'-Dideoxy-3'-thiacytidine (SddC) and 5-fluoro-2',3'-dideoxy-3'-thiacytidine(5-FSddC) were found to be the most potent anti-HBV compounds of those examined. Both compounds resulted in nearly complete cessation of viral DNA replication at 0.5 AM, as monitored by the absence of both intracellular episomal and secreted viral DNAs. The HBVspecific RNAs were not reduced at concentrations that completely blocked HBV DNA replication, suggesting that the inhibitory target is HBV DNA synthesis. The antiviral action of SddC and 5-FSddC was reversible. The concentration of SddC and 5-FSddC required to inhibit 50% of 4-day cell growth in culture was 37 ,uM and more than 200 ,M, respectively. Unlike 2',3'-dideoxycytidine, these two compounds do not affect mitochondrial DNA synthesis in cells at concentrations lower than that required to inhibit cell growth. In view of the potent and selective antiviral activity, both SddC and 5-FSddC should be further evaluated for the treatment of human HBV infection.

PHY906, a four-herb Chinese medicine formula first described 1800 years ago, decreases gastrointestinal toxicity induced by the chemotherapeutic drug CPT-11 (irinotecan), as shown in a phase I/II clinical study. Similarly, in a murine colon 38 allograft model, PHY906 increased the antitumor activity of CPT-11 while decreasing animal weight loss caused by CPT-11. Here, we have further examined the effect of PHY906 on the intestinal toxicity caused by CPT-11 in mice. PHY906 did not protect against the initial DNA damage and apoptosis triggered by CPT-11 in the intestine, but by 4 days after CPT-11 treatment, PHY906 had restored the intestinal epithelium by promoting the regeneration of intestinal progenitor or stem cells and several Wnt signaling components. PHY906 also potentiated Wnt3a activity in human embryonic kidney-293 cells. Furthermore, PHY906 exhibited anti-inflammatory effects in mice by decreasing the infiltration of neutrophils or macrophages, tumor necrosis factor-alpha expression in the intestine, and proinflammatory cytokine concentrations in plasma. Chemical constituents of PHY906 potently inhibited nuclear factor kappaB, cyclooxygenase-2, and inducible nitric oxide synthase. Our results show that the herbal medicine PHY906 can counteract the toxicity of CPT-11 via several mechanisms that act simultaneously.

Ca2؉ signals regulate cell proliferation, but the spatial and temporal specificity of these signals is unknown. Here we use selective buffers of nucleoplasmic or cytoplasmic Ca 2؉ to determine that cell proliferation depends upon Ca 2؉ signals within the nucleus rather than in the cytoplasm. Nuclear Ca 2؉ signals stimulate cell growth rather than inhibit apoptosis and specifically permit cells to advance through early prophase. Selective buffering of nuclear but not cytoplasmic Ca 2؉ signals also impairs growth of tumors in vivo. These findings reveal a major physiological and potential pathophysiological role for nucleoplasmic Ca 2؉ signals and suggest that this information can be used to design novel therapeutic strategies to regulate conditions of abnormal cell growth. Ca2ϩ is a ubiquitous second messenger that mediates a wide range of cellular responses such as contraction, fluid and electrolyte secretion, exocytosis, gene transcription, and apoptosis (1). This ability to simultaneously control multiple processes occurs by careful modulation of Ca 2ϩ signals, not only over time but in different subcellular regions as well (2). For example, polarized Ca 2ϩ waves direct apical secretion in epithelia (3), whereas presynaptic increases in Ca 2ϩ trigger neurotransmitter release (4) and mitochondrial increases in Ca 2ϩ regulate apoptosis (5, 6). Ca 2ϩ signals also can be regulated independently in the nucleoplasm relative to the cytoplasm (7,8), but the physiological significance of this aspect of spatial control is not entirely understood. Nucleoplasmic Ca 2ϩ signals have distinct effects on activation of transcription factors (9, 10) and kinases (11, 12), but it is not known whether nuclear Ca 2ϩ signals also regulate more global aspects of cell function. Because cell proliferation (13,14) and progression through the cell cycle (15, 16) are Ca 2ϩ -dependent, we investigated the relative roles of nuclear and cytoplasmic Ca 2ϩ on cell growth. EXPERIMENTAL PROCEDURESMaterials, Reagents, and Cell Lines-SKHep1, HepG2, and HEK-293 cell lines were obtained from the American Type Culture Collection (Manassas, VA) and were used for all experiments. The cells were grown at 37°C with 5% CO 2 :95% O 2 in Dulbecco's modified Eagle's medium supplemented with 1% penicillin-streptomycin and 10% heat-inactivated fetal bovine serum, all from Invitrogen. The cells were grown on glass coverslips overnight in the absence of serum before infection with each parvalbumin (PV) 2 construct. Generation of Parvalbumin Constructs and AdenoviralInfection-Constructs encoding red fluorescence protein (DsRed) from Clontech (Mountain View, CA) and targeted PV proteins (PV-NLS, PV-NES, and PV-NLS-CD) were PCR-amplified and subcloned into pShuttle-CMV (kindly provided by Bert Vogelstein, Johns Hopkins) by restriction digestion with XhoI and XbaI to generate pShuttle-CMV-PVNLS-DSR, pShuttle-CMV-PVNES-DSR, and pShuttle-CMV-PVNLS-CD-DSR. Recombinant adenoviruses were generated by transformation of pShuttle-CMV-PV-NLS-DSR into AdEasier-1 cells, a deri...

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Herpes simplex virus (HSV) encodes a DNA polymerase that is similar in several respects to the replicative mammalian DNA polymerase a. Recently, these and other DNA polymerases have been shown to share several regions of protein sequence similarity. Despite these similarities, antiviral drugs that mimic natural polymerase substrates specifically inhibit herpesvirus DNA polymerases. To study amino acids involved in substrate and drug recognition, we have characterized and mapped altered drug sensitivity markers of nine HSV pol mutants and sequenced the relevant portions of these mutants. The mutations were found to occur within four relatively small regions. One such region, which we designate region A, has sequence similarity only to DNA polymerases that are sensitive to certain antiviral drugs. The other three regions contain sequences that are similar among various DNA polymerases. The multiple mutations occurring within two of these regions make it likely that the regions interact directly with drugs and substrates. Our results lead us to favor a model in which protein folding allows interactions among the four regions to form the substrate and drug binding sites.DNA polymerases are central enzymes in DNA replication. In prokaryotic systems, studies of DNA polymerases have been greatly aided by model systems to which both genetic and biochemical methods can be applied. The herpes simplex virus (HSV) DNA polymerase, which is essential for HSV replication (1), provides such a model system for the study of eukaryotic DNA polymerases. This enzyme resembles the mammalian replicative DNA polymerase a (pol a) in several respects, including nuclear localization (2) and sensitivities to inhibitors such as aphidicolin (3-7) and dideoxythymidine triphosphate (4). Recently, HSV DNA polymerase (HSV pol), DNA polymerase I of yeast, a variety of animal and bacterial virus DNA polymerases, and pol a were shown to share six regions of striking sequence similarity (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Of the viral polymerases sequenced to date, however, HSV pol appears most closely related to pol a (8). The six regions of sequence similarity have been designated regions I-VI by Wong et al. (8), with region I being most similar among the various polymerases and region VI being the least similar (Fig. 1). Such sequence similarities suggest conservation during evolution and important functional roles for these regions.Despite these similarities, HSV pol is more sensitive than pol a to a number of inhibitors and thus serves as the ultimate target for a number of selective antiviral drugs. These include the pyrophosphate (PP,) analogs phosphonoacetic acid (PAA) and phosphonoformic acid (20, 21), and the triphosphates of nucleoside analogs such as acyclovir (ACV), vidarabine, ganciclovir, and bromovinyldeoxyuridine (BVdU) (22-25). In addition, aphidicolin appears to inhibit HSV pol competitively with certain deoxynucleoside triphosphates (dNTPs) (5-7).The availability of viral pol mutants that exhibit altered se...

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