NMR analyses on N-hydroxymethylated nucleobases - implications for formaldehyde toxicity and nucleic acid demethylases.

Formaldehyde is produced in cells by enzyme-catalysed demethylation reactions, including those occurring on N-methylated nucleic acids. Formaldehyde reacts with nucleobases to form N-hydroxymethylated adducts that may contribute to its toxicity/carcinogenicity when added exogenously, but the chemistry of these reactions has been incompletely defined. We report NMR studies on the reactions of formaldehyde with canonical/modified nucleobases. The results reveal that hydroxymethyl hemiaminals on endocyclic nitrogens, as observed with thymidine and uridine monophosphates, are faster to form than equivalent hemiaminals on exocyclic nitrogens; however, the exocyclic adducts, as formed with adenine, guanine and cytosine, are more stable in solution. Nucleic acid demethylase (FTO)-catalysed hydroxylation of (6-methyl)adenosine results in (6-hydroxymethyl)adenosine as the major observed product; by contrast no evidence for a stable 3-hydroxymethyl adduct was accrued with FTO-catalysed oxidation of (3-methyl)thymidine. Collectively, our results imply N-hydroxymethyled adducts of nucleic acid bases, formed either by reactions with formaldehyde or via demethylase catalysis, have substantially different stabilities, with some being sufficiently stable to have functional roles in disease or the regulation of nucleic acid/nucleobase activity.

TNF blockade: an inflammatory issue.

Tumor necrosis factor (TNF), initially discovered as a result of its antitumor activity, has now been shown to mediate tumor initiation, promotion, and metastasis. In addition, dysregulation of TNF has been implicated in a wide variety of inflammatory diseases including rheumatoid arthritis, Crohn's disease, multiple sclerosis, psoriasis, scleroderma, atopic dermatitis, systemic lupus erythematosus, type II diabetes, atherosclerosis, myocardial infarction, osteoporosis, and autoimmune deficiency disease. TNF, however, is a critical component of effective immune surveillance and is required for proper proliferation and function of NK cells, T cells, B cells, macrophages, and dendritic cells. TNF activity can be blocked, either by using antibodies (Remicade and Humira) or soluble TNF receptor (Enbrel), for the symptoms of arthritis and Crohn's disease to be alleviated, but at the same time, such treatment increases the risk of infections, certain type of cancers, and cardiotoxicity. Thus blockers of TNF that are safe and yet efficacious are urgently needed. Some evidence suggests that while the transmembrane form of TNF has beneficial effects, soluble TNF mediates toxicity. In most cells, TNF mediates its effects through activation of caspases, NF-kappaB, AP-1, c-jun N-terminal kinase, p38 MAPK, and p44/p42 MAPK. Agents that can differentially regulate TNF expression or TNF signaling can be pharmacologically safe and effective therapeutics. Our laboratory has identified numerous such agents from natural sources. These are discussed further in detail.

Diosgenin inhibits osteoclastogenesis, invasion, and proliferation through the downregulation of Akt, I kappa B kinase activation and NF-kappa B-regulated gene expression.

Diosgenin, a steroidal saponin present in fenugreek (Trigonella foenum graecum) and other plants, has been shown to suppress inflammation, inhibit proliferation, and induce apoptosis in a variety of tumor cells, but through a mechanism that is poorly understood. In the present study, we report that diosgenin inhibits receptor-activated nuclear factor-kappaB ligand-induced osteoclastogenesis, suppresses tumor necrosis factor (TNF)-induced invasion, and blocks the proliferation of tumor cells, all activities known to be regulated by NF-kappaB. Diosgenin suppressed TNF-induced NF-kappaB activation as determined by DNA binding, activation of IkappaBalpha kinase, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation through inhibition of Akt activation. NF-kappaB-dependent reporter gene expression was also abrogated by diosgenin. TNF-induced expression of NF-kappaB-regulated gene products involved in cell proliferation (cyclin D1, COX-2, c-myc), antiapoptosis (IAP1, Bcl-2, Bcl-X(L), Bfl-1/A1, TRAF1 and cFLIP), and invasion (MMP-9) were also downregulated by the saponin. Diosgenin also potentiated the apoptosis induced by TNF and chemotherapeutic agents. Overall, our results suggest that diosgenin suppresses proliferation, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression and enhances apoptosis induced by cytokines and chemotherapeutic agents.

Atiprimod blocks STAT3 phosphorylation and induces apoptosis in multiple myeloma cells.

Multiple myeloma (MM) accounts for 1 % of all cancer deaths. Although treated aggressively, almost all myelomas eventually recur and become resistant to treatment. Atiprimod (2-(3-Diethylaminopropyl)-8,8-dipropyl-2-azaspiro[4,5] decane dimaleate) has exerted anti-inflammatory activities and inhibited oeteoclast-induced bone resorption in animal models and been well tolerated in patients with rheumatoid arthritis in phase I clinical trials. Therefore, we investigated its activity in MM cells and its mechanism of action. We found that Atiprimod inhibited proliferation of the myeloma cell lines U266-B1, OCI-MY5, MM-1, and MM-1R in a time- and dose-dependent manner. Atiprimod blocked U266-B1 myeloma cells in the G(0)/G(1) phase, preventing cell cycle progression. Furthermore, Atiprimod inhibited signal transducer and activator of transcription (STAT) 3 activation, blocking the signalling pathway of interleukin-6, which contributes to myeloma cell proliferation and survival, and downregulated the antiapoptotic proteins Bcl-2, Bcl-X(L), and Mcl-1. Incubation of U266-B1 myeloma cells with Atiprimod induced apoptosis through the activation of caspase 3 and subsequent cleavage of the DNA repair enzyme poly(adenosine diphosphate-ribose) polymerase. Finally, Atiprimod suppressed myeloma colony-forming cell proliferation in fresh marrow cells from five patients with newly diagnosed MM in a dose-dependent fashion. These data suggest that Atiprimod has a role in future therapies for MM.

Autocrine lymphotoxin production in Epstein-Barr virus-immortalized B cells: induction via NF-kappaB activation mediated by EBV-derived latent membrane protein 1.

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cells express high levels of lymphotoxin and use this molecule as an autocrine growth factor. We hypothesized that the EBV-derived latent membrane protein 1 (LMP1) mediates lymphotoxin production by inducing NF-kappaB binding to the lymphotoxin promoter. We assessed lymphotoxin production, LMP1 expression, and NF-kappaB activation in Z-43 (EBV-positive lymphoblastoid cells), Daudi (EBV-positive Burkitt's cells), and 3A4 (EBV-negative Burkitt's cells containing a stably transfected tetracycline-inducible LMP1 construct). Z-43 cells expressed high levels of LMP1 (immunoblot) and lymphotoxin (ELISA); the EBV-positive Burkitt's lymphoma line Daudi expressed neither LMP1 nor lymphotoxin. Similarly, induction of LMP1 in the 3A4 cells (exposed to tetracycline) was accompanied by a 13-fold increase in lymphotoxin levels (ELISA) as compared to uninduced (LMP1-negative) cells. EMSAs demonstrated high levels of NF-kappaB activation in Z-43 and tetracycline-induced 3A4 cells, but much lower levels in the uninduced 3A4 cells. Exposure of these cells to Bay 11-7082 (an inhibitor of IkappaB phosphorylation and, therefore, NF-kappaB activation) abrogated NF-kappaB binding and lymphotoxin production in a dose-dependent manner in both Z-43 and 3A4 cells. Therefore, in our model system, autocrine lymphotoxin production is largely driven by NF-kappaB activation, which is in turn mediated by EBV-derived LMP1 signaling.

Involvement of Ras and MAP kinase (ERK-1) in cisplatin-induced activation of murine bone marrow-derived macrophages.

Cisplatin (cis-diamminedichloroplatinum II), a potent antitumor compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the mechanism by which cisplatin activates these cells is poorly characterised. Our earlier findings indicate that cisplatin treatment stimulates rapid tyrosine phosphorylation in a number of cellular proteins in murine macrophages. This initial tyrosine phosphorylation is an important regulatory mechanism and is followed by activation of several other proteins. In the present study, we report the involvement of other key molecules and the role of tyrosine phosphorylation in their activation in the signaling cascade of cisplatin. We observed the involvement of Ras (a low molecular weight GTP-binding protein) and ERK-1 (a MAP kinase) in this signaling cascade. Cisplatin treatment results in an increase in the expression of both Ras and ERK-1 in a dose-dependent manner, which was dependent upon tyrosine phosphorylation. Genistein a PTK inhibitor inhibited the cisplatin induced expression of Ras and ERK-1. These findings indicate that Ras and ERK-1 are important signaling molecules involved in the tumoricidal activation of macrophages with cisplatin and is dependent on initial tyrosine phosphorylation.

Protein kinase C: a potential pathway of macrophage activation with cisplatin.

Cisplatin (CP) has been reported to activate murine macrophages to tumoricidal state, however, its mechanism of action is not known. In the present study it is reported that the production of: (a) interleukin-1 (IL-1); (b) tumor necrosis factor (TNF); (c) nitric oxide (NO); and (d) macrophage-mediated cytotoxicity by cisplatin-treated bone marrow-derived macrophages were inhibited by PKC inhibitors H-7 and chelerythrine chloride. Also, it was observed that treatment of macrophages with CP resulted in the translocation of PKC from the cytosol to the membrane fraction. These findings suggest the involvement of PKC in the activation of bone marrow-derived macrophages with cisplatin.

Expression of LFA-1 adhesion molecules on cisplatin-treated macrophages.

Appropriately activated mononuclear phagocytes mediate contact-dependent tumoricidal activity. Adhesion structures involved in contact-dependent tumor cytotoxicity have not been defined. The present study was aimed at identifying the adhesion structure involved in the tumoricidal activity of cisplatin-activated murine peritoneal macrophages. Tumor cells of different histological origin were used as targets in a 24-h cytotoxicity assay. Anti-CD18 (LFA-1 beta) substantially inhibited macrophage cytotoxicity whereas anti-LFA-1 alpha marginally inhibited macrophage-mediated cytotoxicity. When combined together, almost complete inhibition of tumoricidal activity was observed. Activated macrophages showed augmented binding to target cells and anti-LFA MAb inhibited the binding of resting and activated macrophages to target cells. Cisplatin augmented the expression of LFA-1 alpha and beta integrins and LPS had no effect as assessed by immunoprecipitation. These results implicate that in cisplatin activated macrophages LFA-1 alpha and beta integrins are important molecules in contact-dependent tumoricidal activity.

Cisplatin-induced activation of murine bone marrow-derived macrophages require protein tyrosine phosphorylation.

The aim of the present study is to evaluate the involvement of tyrosine phosphorylation in the signal transduction mechanism of cisplatin-induced macrophage activation in vitro. Stimulation of bone marrow-derived macrophages (BMDM) with cisplatin (CP) resulted in a time- and dose-dependent phosphorylation of several proteins having estimated molecular weights of approximately 18, 20, 21, 30, 33, 35, 39, 41, 44, 58 and 123 kD, detected by immunoblot using anti-phosphotyrosine antibody. CP-induced tyrosine phosphorylation was inhibited by the tyrosine kinase inhibitor genistein. Using this inhibitor, we were able to correlate tyrosine phosphorylation with several functional effects of CP on murine bone marrow-derived macrophages (BMDM). Treatment of macrophages with genistein before incubation with CP completely inhibited the CP-induced tumoricidal activation of macrophages as well as production of TNF and NO, whereas pre-treatment of macrophages with phosphatase inhibitor sodium vanadate upregulated macrophage activation in addition to enhanced protein tyrosine phosphorylation. Taken together, these data suggest that tyrosine phosphorylation play a critical regulatory role in the activation of macrophages with CP.

Cisplatin-stimulated murine bone marrow-derived macrophages secrete oncostatin M.

Cisplatin (CP), a widely used anticancer drug activates cells of the immune system to a tumoricidal state, and thus functions as a potent biological response modifier. Expression of oncostatin M (OSM), a novel cytokine having a growth regulatory effect, was studied in bone marrow-derived macrophages treated with cisplatin. Supernatants from CP-stimulated macrophages were found to be cytostatic for OSM-sensitive A375 melanoma cells. Immunoblot analysis with anti-OSM antibody revealed that expression of OSM in macrophages upon CP stimulation is a rapid process and within 30 min of CP treatment, a significant amount of OSM is secreted into the culture supernatant. These results therefore indicate that CP can stimulate murine bone marrow-derived macrophages to produce OSM which can be implicated as one of the cytostatic/ cytocidal factors in the antitumour action of cisplatin-stimulated macrophages.

Ecosystem studies on upper region of Ganga River, India.

A multi-disciplinary research programme on the Ganga River Ecosystem was launched by the Government of India in 1983 to collect information on its attributes. Monitoring of the initial 509 km unpolluted and unmonitored region of the river falling in partly mountainous and partly upper plain stretches for two years revealed good water quality. The Song River (a tributary) catchment, a victim of extensive mining activity in the past, was found to add maximum mineral load. The Bhagirathi River was found to carry maximum suspended solid load. Organic pollution was low throughout, occasionally showing seasonal and local peaks. The river exhibited a high oxidative state with pH falling in a slightly alkaline range and nutrient levels being very low.Diatoms formed a major part of the encountered genera of phytoplankton. Zooplankton were mainly represented by protozoans. Saprophytic bacteria underwent large spatial and temporal fluctuations. Coliforms exhibited an increasing trend with downstream river distance. The source of pollution could not be specifically characterized from an FC/FS ratio. Only one sample tested positive for enteric virus. The forms of benthic macroinvertebrates indicated a clean stream environment. It was observed that diversity indices, together with evenness and community comparison, could provide a promising approach to determine the state of the community.Eight heavy metals investigated, Cu, Zn, Fe, Cd, Mn, Pb, Ni and Co, were found to be present in the river water and bed sediments. The prominent mode of metal transport was found to be via the suspended load. The concentration of dissolved metals was found within WHO permissible limits. The heavy metal status of the Ganga River was compared with other rivers of the world. Sorptive properties of sediments were found to be similar to the general sorptive behaviour of the clays. Laboratory studies exhibited reasonable short t 90 values for coliform survival in Ganga water. Faecal streptococcus survived longer.