Oral application of a periodontal pathogen impacts SerpinE1 expression and pancreatic islet architecture in prediabetes.

BACKGROUND AND OBJECTIVES: Epidemiological studies suggest a close association between periodontitis and prediabetes/insulin resistance (IR) but whether periodontitis causes prediabetes in humans is not known. Using various animal models, we have recently established that periodontitis can be an initiator of prediabetes, which is characterized by glucose intolerance, hyperinsulinemia and IR. In addition, our in vitro studies indicated that Porphyromonas gingivalis (Pg) induced insulin secretion in MIN6 ß cells and this induction was in part SerpinE1 (plasminogen activator inhibitor 1, PAI1) dependent. However, the mechanism(s) by which periodontitis induces prediabetes is not known. As α and ß cells in pancreatic islets are the major modulators of glucose levels, we investigated whether experimental periodontitis by oral application of a periodontal pathogen caused molecular and/or cellular alterations in pancreatic islets and whether SerpinE1 was involved in this process. MATERIAL AND METHODS: We induced periodontitis in C57BL/6 mice by oral application of a periodontal pathogen, Pg, and determined changes that occurred in islets following 22 weeks of Pg application. Pancreatic islet architecture was determined by 2-D and 3-D immunofluorescence microscopy and SerpinE1 and its target, urokinase plasminogen activator (uPA), as well as insulin, glucagon and Pg/gingipain in islets were detected by immunofluorescence. The presence of apoptotic islet cells was determined by both histochemical and immunofluorescence TUNEL assays. To investigate further the direct effect of Pg on apoptosis and the involvement of SerpinE1 in this process, we used SerpinE1 knockdown and scrambled control clones of the MIN6 pancreatic ß-cell line. RESULTS: Pg/gingipain was detected in both the periodontium and pancreas in the experimental group. Islets from animals that were administered Pg orally (experimental group) developed significant changes in islet architecture, upregulation of SerpinE1, and increased ß-cell apoptosis compared with the control group. We also observed that exposure of MIN6 cells to Pg in vitro resulted in apoptosis. However, apoptosis was significantly reduced when SerpinE1 expression by MIN6 cells was knocked down. CONCLUSION: Oral application of the periodontal pathogen Pg to C57BL/6 mice induces periodontitis, translocation of Pg/gingipain to the pancreas and results in complex alterations in pancreatic islet morphology. SerpinE1 appears to be involved in this process.

A novel anticancer agent ARC antagonizes HIV-1 and HCV.

Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) pose major public health concerns worldwide. HCV is clearly associated with the occurrence of hepatocellular carcinoma, and recently HIV infection has also been linked to the development of a multitude of cancers. Previously, we identified a novel nucleoside analog transcriptional inhibitor ARC (4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide) that exhibited proapoptotic and antiangiogenic properties in vitro. Here, we evaluated the effect of ARC on HIV-1 transcription and HCV replication. Using reporter assays, we found that ARC inhibited HIV-1 Tat-based transactivation in different cell systems. Also, using hepatoma cells that harbor subgenomic and full-length replicons of HCV, we found that ARC inhibited HCV replication. Together, our data indicate that ARC could be a promising candidate for the development of antiviral therapeutics against HIV and HCV.

Differences in mutant p53 protein stability and functional activity in teniposide-sensitive and -resistant human leukemic CEM cells.

We examined p53 protein stability and DNA damage-induced p53-dependent responses in a human leukemic CEM cell line and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5 ( approximately 40 and 400-fold resistant to teniposide, respectively). Although all cell lines contain the same p53 mutations at codons 175 (Arg-->His) and 248 (Arg-->Gln), the constitutive levels of p53 were progressively increased with the resistance of the cells to teniposide. By pulse-chase experiments, we found that the half-lives of mutant p53 protein were approximately 12, 17, and >30 h in CEM, CEM/VM-1, and CEM/VM-1-5 cells, respectively. The prolonged half-lives of p53 in these cells is consistent with the fact that the protein harbors the indicated mutations. Of note, however, is the fact that the increased p53 protein half-lives in the two drug-resistant cell lines corresponds to a proportional decrease in MDM2 protein levels but an increase in p53-MDM2 binding interactions. This suggests that MDM2-mediated p53 degradation may be altered in our leukemic cell lines. The DNA damage-induced p53 response is fully functional in the drug-sensitive CEM cells containing a mutant p53, but this pathway is attenuated in the drug-resistant cells. Specifically, while the mutant p53 was phosphorylated at serine-15 in response to ionizing radiation in all these cell lines, mutant p53 induction in response to teniposide or ionizing radiation and induction of the p53-target genes, p21 and GADD45 only occurred in the drug-sensitive CEM cells. As assessed by MTT cytotoxicity assay, CEM cells were also significantly more sensitive to ionizing radiation, compared to the drug-resistant cell lines, and this correlated with p53 induction. Collectively, these results suggest that changes in constitutive mutant p53 protein levels, p53-MDM2 binding interactions, and altered regulation of the DNA damage-inducible p53-dependent pathway may play a role in drug- and radiation-responsiveness in these cells.

Mechanism of interstrand cross-linking of DNA by anticancer 2-haloethylnitrosoureas.

E and Z-2-haloethyldiazotates, which have been postulated as the ultimate electrophiles responsible for the biological activity of 2-haloethylnitrosoureas (HENUs), have been synthesized and characterized by 15N-nmr. Their stability and solubility in organic solvents are increased by forming crown ether complexes. While E and Z forms are configurationally stable in solution the Z form cyclizes at greater than or equal to -20 degrees C to a 1,2,3-oxadiazoline. The E isomers cross-link DNA, in contrast to the Z isomers. However, both E and Z (2'chloroethyl)thioethyldiazotates (neither of which may cyclize) cross-link DNA extremely efficiently. The cross-linking by these agents is a two-step process and increases with the (G + C) content of the DNA. E-2-chloroethyldiazotate exhibits activity against P388 leukaemia in vivo, lending credence to the suggestion that it is the ultimate electrophile from HENUs. Ab initio calculations predicted the optimized geometry, LUMO energies and atom contributions and the net atomic charges for the diazohydroxides and the HOMO energies and atom contributions for the alternative DNA base sites. An analysis based on Frontier Orbital methods invoking the Hard and Soft Acids and Bases theory permitted an interpretation of the formation of a cross-link site and several modified bases isolated from the reaction of HENUs with DNA.

P-TEFb inhibitors interfere with activation of p53 by DNA-damaging agents.

Tumor suppressor p53 is stabilized in response to gamma-irradiation or treatment with DNA-damaging agents, and as a result p53 transcriptionally activates its targets leading to cell-cycle arrest or apoptosis. P-TEFb (positive transcription elongation factor b) inhibitors such as flavopiridol or 4-amino-6-hydrazino-7-b-d-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide (ARC) upregulate p53 protein levels, but inhibit the expression of its targets p21 and hdm2. DNA-damaging agents, doxorubicin and cisplatin are being used in combination with P-TEFb inhibitor flavopiridol in clinical trials for the treatment of some cancer patients. In this study, we found that P-TEFb inhibitors block the phosphorylation of p53 induced by doxorubicin. Furthermore, treatment of cells with P-TEFb inhibitors together with doxorubicin inhibits doxorubicin-induced binding of p53 to DNA and p53 transcriptional activity. These data suggest that P-TEFb inhibitors may antagonize the activation of p53 by DNA-damaging agents in tumors with wild-type p53.

Proapoptotic compound ARC targets Akt and N-myc in neuroblastoma cells.

We have previously described the identification of a nucleoside analog transcriptional inhibitor ARC (4-amino-6-hydrazino-7-beta-D-ribofuranosyl-7H-pyrrolo[2,3-d]-pyrimidine-5-carboxamide) that was able to induce apoptosis in cancer cell lines of different origin. Here, we report the characterization of ARC on a panel of neuroblastoma cell lines. We found that these cell lines were more than 10-fold sensitive to ARC than to the well-known nucleoside analog DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole), and that ARC-induced apoptosis proceeds through mitochondrial injury. Also, we observed that ARC-mediated cell death was accompanied by caspase-3 cleavage and repression of antiapoptotic proteins such as Mcl-1 and survivin. Conversely, we found that overexpression of Mcl-1-protected neuroblastoma cell line NB-1691 from ARC-induced apoptosis. Furthermore, we found that while ARC inhibited the phosphorylation of Akt Ser-473 in multiple cancer cell lines, forced expression of myristoylated Akt promoted resistance to ARC-induced apoptosis in neuroblastoma cells. In addition, we observed that ARC was able to downregulate the protein levels of N-myc, a commonly amplified oncogene in neuroblastomas, and Akt protected N-myc from ARC-induced downregulation. These data suggest that ARC may antagonize different antiapoptotic pathways and induce apoptosis in neuroblastoma cells via multiple mechanisms. Overall, ARC could represent an attractive candidate for anticancer drug development against neuroblastomas.

Cytotoxic signalling by inhibitors of DNA topoisomerase II.

DNA topoisomerase (topo) II inhibitors either stabilize DNA-topo II complexes by blocking DNA religation (e.g. etoposide) or block the enzyme's catalytic activity (e.g. dexrazoxane). The former class of drugs causes direct DNA damage through topo II, while the latter class does not, but both classes cause apoptosis. We cloned the Fas ligand (FasL) promoter and coupled it to the luciferase gene. Treatment of cells transfected with this construct revealed that complex-stabilizing (DNA-damaging) agents induce FasL expression, but the catalytic inhibitors do not, suggesting that the FasL pathway may not be involved in all cases of topoisomerase-mediated apoptosis. Some topo II inhibitors activate a pathway involving stress-activated protein kinases, which include c-Jun N-terminal kinase-1 (JNK-1). We will discuss the effects of these agents on components of this pathway. Our earlier work revealed that topo IIalpha interacts with the cell cycle regulatory protein, retinoblastoma protein (Rb). This interaction and the subcellular distribution of these proteins are altered by topo II inhibitory drugs and lead to apoptosis. In addition, agents that affect Rb, such as E1A and E2F1/DP-1, when transfected into cells, also alter topo IIalpha-Rb localization, activate jun kinase pathways and cause apoptosis. This paper discusses current studies that are designed to determine the contributions of these signalling events to the alterations in subcellular protein distribution and apoptosis. We suggest that protein-protein interactions are important for mediation of cytotoxic signalling by anticancer drugs.

Functional interaction between human topoisomerase IIalpha and retinoblastoma protein.

DNA topoisomerase II-an essential nuclear enzyme in DNA replication and transcription, chromatin segregation, and cell cycle progression-is also a target of clinically useful anticancer drugs. Preliminary observations of a positive correlation between the expression of topoisomerase (topo) IIalpha and the retinoblastoma protein (Rb) in a series of rhabdomyosarcoma cells prompted us to ask whether these two proteins interact in vivo. Using human rhabdomyosarcoma and leukemic cell lines, we found a physical association between topo IIalpha and Rb protein by reciprocal immunoprecipitation and immunoblotting, in which topo IIalpha appeared to interact primarily with the underphosphorylated form of Rb. Experiments with truncated glutathione S-transferase-Rb fusion proteins and nuclear extracts of Rh1 rhabdomyosarcoma cells indicated that topo IIalpha binds avidly to the A/B pocket domain of Rb, which contains the intact spacer amino acid sequence. To determine whether this interaction has functional consequences in vivo, we expressed wild-type and mutant Rb in human cervical carcinoma cells lacking functional Rb. Wild-type, but not mutant, Rb inhibited topo II activity in nuclear extracts of these transfected cells. Moreover, purified wild-type Rb inhibited the activity of purified human topo IIalpha, indicating a direct interaction between these two proteins. We conclude that topo IIalpha associates physically with Rb in interactions that appear to have functional significance.

A structure-function relationship among reserpine and yohimbine analogues in their ability to increase expression of mdr1 and P-glycoprotein in a human colon carcinoma cell line.

We previously showed that there is a structure-function relationship among reserpine and yohimbine analogues in their ability to inhibit the function of P-glycoprotein (P-gp) and reverse multidrug resistance (MDR). Because some P-gp inhibitors (e.g., verapamil and nifedipine) can increase mdr1 and P-gp expression in human colon carcinoma cell lines, we used our reserpine/yohimbine analogues to determine whether there was a structural requirement for this induction. We found that 10 microM reserpine increased both mdr1 and P-gp expression by 4-10-fold in 48 hr in a human colon carcinoma cell line that expresses moderate levels of mdr1 (LS180-Ad50) but not in several other cell lines that expressed no mdr1. The reserpine/yohimbine analogues rescinnamine, trimethoxybenzoylyohimbine, and LY191401 (compound G), all of which contain the three structural elements used to describe the MDR pharmacophore, also increased both mdr1 and P-gp expression significantly. Despite some exceptions, we found that there was a good association between the ability of these analogues to induce mdr1 and P-gp expression and their ability to reverse vinblastine and doxorubicin resistance, revealing a structure-function relationship for this phenomenon. The increased P-gp expressed by these cells appeared to be functional, as determined by flow cytometric detection of rhodamine 123 retention. The increased expression was suppressed by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an RNA synthesis inhibitor, whereas the protein synthesis inhibitor cycloheximide enhanced the expression several-fold, suggesting that induction of mdr1 by these analogues is regulated at both the transcriptional and post-transcriptional levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular recognition between oligopeptides and nucleic acids: novel imidazole-containing oligopeptides related to netropsin that exhibit altered DNA sequence specificity.

Oligopeptides have been synthesized that are structurally related to the antiviral antitumor antibiotic netropsin, but in which each of the pyrrole units is successively replaced by an imidazole moiety, as well as their di- and triimidazole-containing counterparts. These compounds bind to duplex DNA with constants in the range (1.06-1.98) X 10(6) M-1 but not to single-stranded DNA. Since they bind to T4 DNA, it is inferred that, like the parent antibiotic netropsin, they are also minor groove selective. This series of compounds exhibits a progressively decreasing preference for AT sites in binding studies with both native DNAs and synthetic oligonucleotides and a corresponding increasing acceptance of GC base pairs. Footprinting experiments utilizing a 139 base pair HindIII/NciI restriction fragment from pBR 322 DNA revealed that these lexitropsins, or information-reading oligopeptides, recognize more sites than the parent netropsin. In addition, some regions of enhanced nuclease action as the result of drug binding to the fragment were identified. The diimidazole compound in particular recognizes GC-rich sites, implying the formation of new hydrogen bonds between G-C(2)NH2 in the minor groove and the additional N3 imidazole nitrogens. It is clear however that, since the lexitropsins appear to tolerate the original (AT)4 site, an N-methylimidazole group on the ligand will permit either a GC or AT base pair in the binding sequence. Another factor that may be significant in molecular recognition is the high negative electrostatic potential of A X T regions of the minor groove, which is likely to strongly influence binding of these cationic species to DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of electrophiles from 2-haloethylnitrosoureas forming cytotoxic DNA cross-links and cyclic nucleotide adducts and the analysis of base site-selectivity by ab initio calculations.

E- and Z-2-haloethyldiazotates--electrophilic species hitherto suggested as intermediates in the reactions of 2-haloethylnitrosoureas (HENUs) under physiological conditions--were synthesized and characterized by 1H-, 15N- and 13C-NMR (nuclear magnetic resonance). They were stabilized and solubilized in organic solvents as their 18-crown-6 ether complexes. Characterization of the Z-2-fluoroethyldiazotate by 19F- and 13C-NMR, and comparison with the Z-2-chloroethyl compound, confirmed facile cyclization to the 1,2,3-oxadiazoline and subsequent decomposition to nitrogen and ethylene oxide. The E-2-haloethyldiazotates form DNA interstrand cross-links at a rate, and to an extent, and with a DNA base dependence, which parallels the behaviour of the parent HENUs, while the Z isomers alkylate DNA but show minimal cross-linking. Both E-and Z-(2'-chloroethyl)thioethyldiazotates, neither of which can undergo cyclization, cross-link DNA efficiently. Self-consistent-field (SCF) ab initio calculations provided optimized geometries, atomic charges and LUMO (Lowest Unoccupied Molecular Orbital) atom contributions for the E- and Z-2-haloethyldiazohydroxides. The HSAB (Hard and Soft Acids and Bases) theory, in conjunction with HOMO (Highest Occupied Molecular Orbital) values on key DNA base sites, accounted for the observed site-selectivity in the formation of identified cross-links produced by 1,3-bis-(2-chloroethyl)-1-nitrosourea. Independent chemical studies on cytosine derivatives corroborated the predicted site selectivity of attack by electrophiles and the formation of ethanocytidine cyclic adducts.