Bortezomib interacts synergistically with belinostat in human acute myeloid leukaemia and acute lymphoblastic leukaemia cells in association with perturbations in NF-κB and Bim

Interactions between the histone deacetylase inhibitor belinostat and the proteasome inhibitor bortezomib were investigated in acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) cells. Co‐administration of sub‐micromolar concentrations of belinostat with low nanomolar concentrations of bortezomib sharply increased apoptosis in both AML and ALL cell lines and primary blasts. Synergistic interactions were associated with interruption of both canonical and non‐canonical nuclear factor (NF)‐κB signalling pathways, e.g. accumulation of the phosphorylated (S32/S36) form of IκBα, diminished belinostat‐mediated RelA/p65 hyperacetylation (K310), and reduced processing of p100 into p52. These events were accompanied by down‐regulation of NF‐κB‐dependent pro‐survival proteins (e.g. XIAP, Bcl‐xL). Moreover, belinostat/bortezomib co‐exposure induced up‐regulation of the BH3‐only pro‐death protein Bim. Significantly, shRNA knock‐down of Bim substantially reduced the lethality of belinostat/bortezomib regimens. Administration of belinostat ± bortezomib also induced hyperacetylation (K40) of α‐tubulin, indicating histone deacetylase inhibitor 6 inhibition. Finally, in contrast to the pronounced lethality of belinostat/bortezomib toward primary leukaemia blasts, equivalent treatment was relatively non‐toxic to normal CD34⁺ cells. Together, these findings indicate that belinostat and bortezomib interact synergistically in both cultured and primary AML and ALL cells, and raise the possibilities that up‐regulation of Bim and interference with NF‐κB pathways contribute to this phenomenon. They also suggest that combined belinostat/bortezomib regimens warrant further attention in acute leukaemias.     

CaMKIIγ regulates the viability and self-renewal of acute myeloid leukaemia stem-like cells by the Alox5/NF-κB pathway

Acute myeloid leukaemia (AML) is a frequently fatal malignant disease of haematopoietic stem and progenitor cells. The molecular and phenotypic characteristics of AML are highly heterogeneous. Our previous study concluded that CaMKIIγ was the trigger of chronic myeloid leukaemia progression from the chronic phase to blast crisis, but how CaMKIIγ influences AML stem-like cells remains elusive. In this study, we found that CaMKIIγ was overexpressed in AML patients and AML cell lines, as measured by qRT-PCR and Western blot assays. Moreover, CaMKIIγ decreased when the disease was in remission. Using an shRNA lentivirus expression system, we established CaMKIIγ stable-knockdown AML cell lines and found that knockdown of CaMKIIγ inhibited the viability and self-renewal of AML stem-like cell lines. Additionally, the ratio of CD₃₄⁺ AML cell lines decreased, and CaMKIIγ knockdown induced the downregulation of Alox5 levels. We further detected downstream molecules of the Alox5/NF-κB pathway and found that c-myc and p-IκBα decreased while total IκBα remained normal. In conclusion, our study describes a new role for CaMKIIγ as a stem-like cell marker that is highly regulated by the Alox5/NF-κB pathway in AML stem-like cells. CaMKIIγ can participate in the viability and self-renewal of AML stem-like cells by regulating the Alox5/NF-κB pathway.     

Interferonγ and tumour necrosis factorα induce a synergistic antiproliferative response in human Ewing's sarcoma cells in vitro

Three human cell lines derived from Ewing's sarcoma (RM-82, VH-64, and WE-68) were investigated to establish the influence of recombinant human interferon (rhIFN) and tumour necrosis factor (rhTNF) on cell proliferation and survival and to characterize IFN and TNF receptor expression. Incorporation of [³H]thymidine into cells was inhibited by rhIFN after 24 h of incubation. Half-maximal inhibition was observed with 10–80 U/ml rhIFN. A maximal effect (50%–70% inhibition of cell proliferation) was achieved by treatment of cells with 250 U/ml rhIFN. The influence of rhTNF on proliferation was found to differ among cell lines and varied with the concentration and the duration of exposure of cells to this cytokine. In WE-68 and VH-64 cells [³H]thymidine incorporation was not affected by rhTNF up to 2000 U/ml after 96 h of incubation, where-as in RM-82 cells the incorporation was inhibited by 35% after 48 h of incubation with 100 U/ml rhTNF. However, all cell lines showed a synergistic antiproliferative response to the combination of rhIFN and rhTNF after 24 h of incubation. The human recombinant cytokines interleukin(IL)-1, IL-1, IL-2, IL-3, IL-4, IL-6 and granulocyte/macrophagecolony-stimulating factor, tested alone and in combination with rhIFN and rhTNF, had no influence on cell proliferation. Binding studies in the cell lines with¹²⁵I-rhIFN revealed a dissociation constant (K _d ) of 160–306 pM and approximately 8000–13500 receptors/cell. Binding experiments with¹²⁵I-rhTNF indicated 430–1250 receptors/cell withK _d ranging from 13 pM to 162 pM. These data indicate that, among various cytokines, only IFN and TNF are capable of potently reducing Ewing's sarcoma cell growth in vitro. Our data suggest that IFN alone or in combination with TNF may be useful in the design of novel strategies in Ewing's sarcoma therapy.     

IL-1β and IFN-γ induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells,and in islets from pre-diabetic NOD mice

AIMS/HYPOTHESIS: Cytokines and chemokines are important mediators of immune responses due to their ability to recruit and activate leukocytes. Using microarray analysis we observed that rat beta cells exposed to IL-1beta and IFN-gamma have increased mRNA levels of chemokines and IL-15. The aim of this study was to characterize the expression of IP-10, MIP-3alpha, fractalkine and IL-15 in rat beta cells, human pancreatic islets, and in islets isolated from NOD mice, both during the pre-diabetic period and following islet transplantation. METHODS: FACS-purified rat beta cells and human islets were cultured with IL-1beta, IFN-gamma and/or TNF-alpha. Islets were isolated from NOD or BALB/c mice at different ages. For syngeneic islet transplantation, 2- or 3-week-old NOD islets were grafted under the kidney capsule of spontaneously diabetic NOD recipients. Chemokine and IL-15 mRNA expression and protein release were evaluated, respectively, by RT-PCR and ELISA. RESULTS: Human islets and rat beta cells express IP-10, MIP-3alpha, fractalkine and IL-15 mRNAs upon exposure to cytokines. The expression of IL-15, IP-10 and fractalkine is regulated by IFN-gamma, while the expression of MIP-3alpha is IL-1beta-dependent. Moreover, cytokines induced IL-15, IP-10, Mig, I-TAC and MIP-3alpha protein accumulation in culture medium from human islets. In vivo, there was an age-related increase in IL-15, IP-10 and MIP-3alpha expression in islets isolated from NOD mice. Following syngeneic islet transplantation, increased expression of IL-1beta, IFN-gamma, fractalkine, IP-10, MCP-1 and MIP-3alpha mRNAs were observed in the grafts. CONCLUSION/INTERPRETATION: Cytokine-exposed islets or beta cells express chemokines and IL-15. This could contribute to the recruitment and activation of mononuclear cells and development of insulitis in early Type 1 diabetes and during graft destruction.     

Error-free replicative bypass of thymine glycol by the combined action of DNA polymerases κ and ζ in human cells

Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5′ base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Polκ and Polζ function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Polκ at the insertion step and of Polζ at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.     

Hypoxia abrogates antichlamydial properties of IFN-γ in human fallopian tube cells in vitro and ex vivo

IFN-γ has an important role in the adaptive immune response against intracellular pathogens. In urogenital tract (UGT) infections with the obligate intracellular pathogen Chlamydia trachomatis, IFN-γ-mediated control of chlamydial growth implies the JAK-STAT signaling cascades and subsequent induction of the indoleamine 2,3-dioxygenase (IDO). As oxygen concentrations in the UGT are low under physiological conditions (O(2) < 5%) and further decrease during an inflammatory process, we wondered whether antibacterial properties of IFN-γ are maintained under hypoxic conditions. Using primary cells that were isolated from human fallopian tubes and an ex vivo human fallopian tube model (HFTM), we found that even high IFN-γ concentrations (200 units/mL) were not sufficient to limit growth of C. trachomatis under hypoxia. Reduced antibacterial activity of IFN-γ under hypoxia was restricted to the urogenital serovars D and L(2), but was not observed with the ocular serovar A. Impaired effectiveness of IFN-γ on chlamydial growth under hypoxia was accompanied by reduced phosphorylation of Stat-1 on Tyr701 and diminished IDO activity. This study shows that IFN-γ effector functions on intracellular C. trachomatis depend on the environmental oxygen supply, which could explain inadequate bacterial clearance and subsequent chronic infections eventually occurring in the UGT of women.     

Roles of Dermcidin,Salusin-α, Salusin-β and TNF-α in the Pathogenesis of Human Brucellosis

Background:Brucella spp. are facultative intracellular pathogens that can cause chronic infections in many tissues and organs. Objectives: To investigate serum dermcidin, salusin-alpha, salusin-beta and TNF-alpha levels and their correlation with each other in patients with acute brucellosis. Methods: From 50 patients hospitalized upon diagnosis of acute brucellosis, blood samples were collected and dermcidin, salusin-alpha, salusin-beta and TNF-alpha levels in serum samples were measured using an ELISA assay. The control group included 40 volunteers. Results: Brucellosis group had significantly lower plasma dermcidin, salusin- alpha, salusin-beta levels compared to the healthy control group (respectively p:0.008, p<0.001, p<0.001). Moreover, Brucellosis group had significantly higher plasma TNF-alpha levels comparisons with the controls (p=0.002). In the examination of the correlation between TNF-alpha and dermcidin, salusin-alpha and salusin-beta in the brucellosis group, only a negative correlation was found between salusin-beta and TNF-alpha. In the control group, there was a positive and statistically significant correlation between salusin-beta and TNF-alpha. Conclusion: Dermcidin, salusin-alpha, and, particularly salusin-beta levels are important in Brucella pathogenesis. The paradoxical correlation between TNF-alpha and salusin-beta in patients with brucellosis and control group is remarkable. However, there is a need for extensive studies conducted with more patients to further elucidate this topic.     

Effect of 17β-oestradiol on expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar cells

Oestradiol (E(2)) alters lymphocyte functionin vitro including T cell DNA synthesis and B cell immunoglobulin production in human tonsillar, splenic and peripheral blood cells. We have investigated whether one mechanism for this effect is that E(2) modifies the expression of IL-2, IL-6 and IFN-γ mRNA in human tonsillar mononuclear cells. Without E(2), addition of PHA (1 μg ml(-1)) for 10 h increased the expression of IL-2 and IL-6 mRNA but had no significant effect on IFN-γ mRNA. In separated T cells after 24 h incubation, E(2) (7×10(-8) M: ) increased only the IFN-γ mRNA levels. However, when E(2) was present in PHA-stimulated T cell cultures, mRNA levels from all cytokines were suppressed. E(2) decreased IL-2 mRNA levels in the T cell preparation after 24 h culture. For IL-6, E(2) decreased mRNA both in mononuclear cells and T cells after 10 h incubation. For IFN-γ, E(2) decreased mRNA levels in the mononuclear cell preparation after 24 h culture. Stimulation of the T cell preparation with PHA after 24 h incubation with E(2) decreased the IFN-γ mRNA levels compared to the cultures incubated with E(2) only. One part of the action of E(2) may be through a block in the up-regulation of the mRNA of IL-2, IL-6 and IFN-γ in activated cells.     

Characterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis

Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-γ production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection (LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TST+) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120?h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144?h and the percentage of CD4(+) and CD8(+)IFN-γ(+) T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4(+) and CD8(+) precursor cells, as well as decreased number of CD4(+)IFN-γ(+) cells in response to all antigens, whereas CD8(+)IFN-γ(+) cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-γ responses, irrespective of the antigen employed. The CD4(+)/CD8(+) cell ratios showed that in response to PPD CD4(+) precursor and IFN-γ-producer cells are more frequent than their CD8(+) counterparts, and that PTB have a decreased CD4(+)IFN-γ(+)/CD8(+)IFN-γ(+) ratio in response to PPD, CFP-10, and ESAT-6. CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells exhibited a central memory phenotype (CD45RO(+)CD27(+)), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4(+) and CD8(+) precursor cells and CD4(+)IFN-γ(+). HHCs exhibited the highest frequency of CD4(+) and CD8(+) precursors and CD4(+)IFN-γ(+)-producing cells.     

Inhibition of PPARγ in myeloid-lineage cells induces systemic inflammation, immunosuppression, and tumorigenesis

Peroxisome proliferator-activated receptor-γ (PPARγ) is an anti-inflammatory molecule. To study its biologic function in myeloid cells, dominant-negative PPARγ (dnPPARγ) was overexpressed in a myeloid-specific bitransgenic mouse model. In this bitransgenic system, overexpression of the dnPPARγ-Flag fusion protein in myeloid-lineage cells abnormally elevated frequencies and total numbers of IL-7Rα(-)Lin(-)c-Kit(+)Sca-1(-), Lin(-)/Scal(+)/c-Kit(+), common myeloid, and granulocyte-monocyte progenitor populations in the BM. dnPPARγ overexpression led to up-regulation of IL-1β, IL-6, and TNFα in the blood plasma. As a result, CD11b(+)Ly6G(+) cells were systemically increased in association with activation of Stat3, NF-κB, Erk1/2, and p38 molecules. Myeloid-derived suppressor cells (MDSCs) inhibited the proliferation and lymphokine production of wild-type CD4+ T cells in vitro. CD4+ T cells from doxycycline-treated bitransgenic mice displayed reduced proliferation and lymphokine release. Both CD4+ and CD8+ T-cell populations were decreased in doxycycline-treated bitransgenic mice. Multiple forms of carcinoma and sarcoma in the lung, liver, spleen, and lymph nodes were observed in doxycycline-treated bitransgenic mice. BM transplantation revealed that a myeloid-autonomous defect was responsible for MDSC expansion, immunosuppression, and tumorigenesis in these mice. These studies suggest that anti-inflammatory PPARγ in myeloid-lineage cells plays a key role in controlling pro-inflammatory cytokine synthesis, MDSC expansion, immunosuppression, and the development of cancer.     

Influence of acid and bile acid on ERK activity, PPARγ expression and cell proliferation in normal human esophageal epithelial cells

AIM: To observe the effects of acid and bile acid exposure on cell proliferation and the expression of extracellular signal-regulated protein kinase (ERK) and peroxisome proliferator-activated receptor gamma (PPARgamma) in normal human esophageal epithelial cells in vitro. METHODS: In vitro cultured normal human esophageal epithelial cells were exposed to acidic media (pH 4.0-6.5), media containing different bile acid (250 mumol/L), media containing acid and bile acid, respectively. Cell proliferation was assessed using MTT and flow cytometry. The expressions of phosphorylated ERK(1/2) and PPARgamma protein were determined by the immunoblotting technique. RESULTS: Acid-exposed (3 min) esophageal cells exhibited a significant increase in proliferation ratio, S phase of the cell cycle (P<0.05) and the level of phosphorylated ERK(1/2) protein. When the acid-exposure period exceeded 6 min, we observed a decrease in proliferation ratio and S phase of the cell cycle, with an increased apoptosis ratio (P<0.05). Bile acid exposure (3-12 min) also produced an increase in proliferation ratio, S phase of the cell cycle (P<0.05) and phosphorylated ERK(1/2) expression. On the contrary, deoxycholic acid (DCA) exposure (>20 min) decreased proliferation ratio. Compared with bile acid exposure (pH 7.4), bile acid exposure (pH 6.5, 4) significantly decreased proliferation ratio (P<0.05). There was no expression of PPARgamma in normal human esophageal epithelial cells. CONCLUSION: The rapid stimuli of acid or bile acid increase proliferation in normal human esophageal epithelial cells by activating the ERK pathway.     

The role of α-, δ- and F cells in insulin secretion and action

Nowadays, most of the basic pancreatic islet research is focused on the beta and alpha cells. Nonetheless, the pancreatic islets are complete organs with five, up to now, different cell types and dedicated vasculature and neural supply. Every cell contributes in the integration of the islet function and deserves a closer lookup in the saga of the glucose homeostasis. In this brief report we describe the current concepts about the contribution of the non-beta cells in the overall islet contribution to glucose metabolism.     

Inhibition of K562 cell growth and tumor angiogenesis in nude mice by transfection of anti-VEGF hairpin ribozyme gene into the cells

Objective To explore the effect of anti-VEGF hairpin ribozyme gene on the tumor cell growth and tumor angiogenesis in nude mice. Methods The recombinant eukaryotic expression plasmid pcDNA-RZ containing anti-VEGF hairpin ribozyme gene and the empty vector plasmid pcDNA were introduced separately into K562 cells     

Interleukin-1β and interleukin-1α stimulate the N-acetyl-β-glucosaminidase activity of human synovial cells

Summary The properties of synovial cells are altered in vitro by monocyte-macrophage polypeptides (monokines), and these changes could explain some of the properties of the inflamed synovium in rheumatoid disease. Purified monokines have become available only recently for testing on the target synovial cells. We report here that purified human interleukin (IL)-1 and recombinant human Il-1 stimulate the extracellular activity of the lysosomal hydrolase, N-acetyl--glucosaminidase (NAG), of human synovial fibroblast-like cells. In contrast, another monokine, synovial activator, does not increase the NAG activity. Thus NAG is another cellular activity which can be modulated by interleukin-1.