Downregulated SOCS1 expression activates the JAK1/STAT1 pathway and promotes polarization of macrophages into M1 type.

Macrophage polarization is flexible, and involves in different signaling pathways and various transcription factors. Suppressor of cytokine signaling (SOCS) is an important inhibitor of cytokine signaling pathways and also a key physiological regulator for natural and acquired immunity systems. Following transfection of SOCS1 short hairpin (sh)RNA into mouse macrophage cells, reverse transcription­quantitative polymerase chain reaction demonstrated that the mRNA levels of Janus kinase (JAK)1 and signal transducer and activator of transcription (STAT)1 increased significantly. In addition, western blotting indicated that JAK1, STAT1 and p­STAT1 expression was significantly enhanced. Fludarabine can inhibit phosphorylation of STAT1 and SOCS1 expression. When fludarabine was added and SOCS1 shRNA was transfected, the inhibition of fludarabine was weakened, and p­STAT1 expression was upregulated. Flow cytometry detection indicated that, following the downregulation of SOCS1 expression, M1­type cells significantly increased, but the proportion of M2­type cells did not change significantly. Fludarabine can reduce the effect of SOCS1 shRNA on promoting M1­type cell polarization, and macrophages can polarize into both M1 and M2 phenotypes. Further ELISA results presented that, when downregulating SOCS1 expression, interleukin (IL)­4 and IL­10 expression was both downregulated, and tumor necrosis factor (TNF)­α and interferon (IFN)­Î³ expression was significantly upregulated. When adding fludarabine or injecting with the traditional Chinese medicine Xuebijing, IL­4 and IL­10 expression was both significantly upregulated, and TNF­α and IFN­Î³ expression was significantly downregulated. When adding fludarabine and downregulating SOCS1, IL­4, IL­10, TNF­α and IFN­Î³ expression presented no significant changes. The above results indicated that, when SOCS1 expression is downregulated, it will activate the JAK1/STAT1 pathway, and thereby promote the polarization of macrophages into M1 type. The findings are of great importance for understanding occurrence, development and treatment of various immune­related diseases.

Rational design of inhibitors of VirA-VirG two-component signal transduction.

VirA-VirG two-component system regulates the vir (virulence) operon in response to specific host factors (xenognosins) in the plant pathogen Agrobacterium tumefaciens. Using whole cell assays, stable inhibitors inspired by the labile natural benzoxazinone inhibitor HDMBOA are developed. It is found that aromatic aldehydes represent a minimal structural unit for activity. In particular, 3-hydroxy-4,6-dimethoxy-3H-isobenzofuran-1-one (HDI) was found to have the highest activity, making it the most potent developed inhibitor of virulence gene expression in Agrobacterium.

Triggering of CD40 antigen inhibits fludarabine-induced apoptosis in B chronic lymphocytic leukemia cells.

We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean +/- SE: 28.5 +/- 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% +/- 7.8% on average (P = .0077). Because the CD40 antigen activates NF-kappaB/Rel transcription factors in B cells, and NF-kappaB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-kappaB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-kappaB/Rel activity; p50, RelA, and c-Rel components of the NF-kappaB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-kappaB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-kappaB/Rel levels. To determine the involvement of NF-kappaB/Rel activity in the G28-5-mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-kappaB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-kappaB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-kappaB/Rel inhibitors, could improve the therapeutic effect of fludarabine.

Basic fibroblast growth factor (bFGF) upregulates the expression of bcl-2 in B cell chronic lymphocytic leukemia cell lines resulting in delaying apoptosis.

Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine which has recently been shown to delay fludarabine-induced apoptosis in B cell chronic lymphocytic leukemia (B-CLL) cells. To investigate the potential mechanism of bFGF-mediated delay of apoptosis, two EBV-transformed B prolymphocytic cell lines (JVM-2, JVM-13), one EBV-transformed B-CLL cell line (I83CLL), and one non-EBV-transformed B-CLL cell line (WSU-CLL) were used as a model for chronic lymphoid malignancies. Viability data of cells treated with fludarabine alone or in combination with bFGF demonstrated that the addition of bFGF to the cells resulted in prolonged survival. Quantitative assessment of apoptosis-associated DNA strand breaks by in situ TdT labeling showed a protective effect of bFGF on fludarabine-treated cells. The potential effect of bFGF on bcl-2 mRNA expression was analyzed by Northern blotting. Stimulation with bFGF led to a time-dependent accumulation of bcl-2 specific mRNA in all three cell lines. Maximal levels of bcl-2 mRNA expression were detected after 8 h in JVM-2, and after 18 h in JVM-13 and I83CLL. Intracellular bcl-2 protein was also found to be increased upon bFGF stimulation in both EBV- and non-EBV-transformed cells. In addition, exposure of cells from three patients with B-CLL to bFGF showed an upregulation of bcl-2 protein after 4-8 h. Our data demonstrate that bFGF upregulates the expression of bcl-2 in these cells, suggesting that this increase in bcl-2 expression may play a role in the delay of fludarabine-induced apoptosis.

Inhibition of fludarabine metabolism by arabinosylcytosine during therapy.

The active 5'-triphosphate of arabinosyl-2-fluoroadenine (F-ara-ATP) increases the anabolism of arabinosylcytosine (ara-C), whereas ara-C 5'-triphosphate inhibits the phosphorylation of arabinosyl-2-fluoroadenine (F-ara-A) in human leukemia cells in vitro. These interactions have a potential impact on drug scheduling. Clinical trials of relapsed leukemia in which fludarabine (F-ara-A 5'-monophosphate) and ara-C were given in sequence provided the opportunity to evaluate the effects of ara-C infusion on two sequelae: the pharmacokinetics of F-ara-A in plasma and that of F-ara-ATP in leukemia cells. First, F-ara-A pharmacokinetics were altered by ara-C infusion. This was visualized as a transient increase in F-ara-A plasma levels during the ara-C infusion that was given 4 h after fludarabine. The perturbation in F-ara-A plasma levels was dependent on the dose ara-C. Second, peak F-ara-ATP concentrations were lower in leukemia cells of patients who received ara-C in addition to fludarabine as compared with those who received fludarabine alone. The terminal half-life of F-ara-A in plasma and the half-life of intracellular F-ara-ATP were reduced after the ara-C infusion in a concentration-dependent manner. Studies using purified deoxycytidine kinase support the conclusion that the increase in plasma levels of F-ara-A is in part the result of an effective competition by ara-C for phosphorylation by this enzyme, leading to a perturbation of the pharmacokinetics of intracellular F-ara-ATP.

Deoxyadenosine antagonism of the antiviral activity of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosylhypoxanthine.

Deoxyadenosine but not adenosine reversed the antiviral activity of 9-beta-D-arabinofuranosyladenine (ara-A) and 9-beta-D-arabinofuranosylhypoxanthine (ara-H) when used in the presence of coformycin, an inhibitor of adenosine deaminase. In suspension cultures of KB cells, 10 muM ara-A inhibited the replication of herpes simplex virus type 1 by 80%. Concomitant addition of 50 muM deoxyadenosine reduced the antiviral activity of 10 muM ara-A to only 40% inhibition. Adenosine failed to antagonize the antiviral activity. In monolayer cultures of KB cells, the 50% inhibitory concentration of ara-A was increased from 1.5 to 2.9 muM by 2 muM deoxyadenosine and to 8.5 muM by 10 muM deoxyadenosine. Analysis of the dose-response data by a double reciprocal plot method indicated that the antagonism was competitive. The antiviral activity of ara-H also was antagonized by deoxyadenosine. The 50% inhibitory concentration of ara-H was increased from 42 muM to 70, 91, or 121 muM by the concurrent addition of 5, 10, or 20 muM deoxyadenosine. Competitive antagonism could not be demonstrated. In the absence of the adenosine deaminase inhibitor, neither ara-A nor ara-H was antagonized by deoxyadenosine. Since such inhibitors were not available unitl recently, previous investigators were unable to observe the antagonistic capacity of deoxyadenosine.

Adenosine aminohydrolase from monkey brain: partial purification and some kinetic properties.

Adenosine aminohydrolase from monkey brain was purified ten fold. In pH 7.3 phosphate buffer at 37 degrees, this enzyme preparation deaminated adenosine and arabinosyladenine with apparent values for the Michaelis constant of 32 muM and 370 muM respectively. The products of both deamination reactions, i.e., inosine and arabinosylhypoxanthine, were competitive inhibitors with Ki equal to 220 muM and 1,000 muM, respectively. N6-benzyladenosine and 9-(1-hydroxy-2-octyl)adenine were competitive inhibitors and were more effective in inhibiting deamination of arabinosyladenine than of adenosine. It is suggested that these compounds might potentiate arabinosyladenine chemotherapy of neoplasms of the central nervous system.