Normal Peripheral Blood Lymphocytes Research Articles

Abnormal adenosine and dopamine receptor expression in lymphocytes of Lesch–Nyhan patients

Self-injurious behavior is the most outstanding feature of Lesch–Nyhan syndrome and has recently been ascribed to an obsessive–compulsive behavior. Lesch–Nyhan syndrome results from the complete enzyme deficiency of hypoxanthine–guanine phosphoribosyl transferase (HPRT) but the link between abnormal purine metabolism and its neurological and behavioral manifestations remains largely unknown. Previous studies led us to hypothesize that adenosine and dopamine receptor expression could be altered in HPRT-deficient cells. To test this hypothesis, we examined mRNA expressions of adenosine (ADORA2A and ADORA2B) and dopamine receptors (DRD1 and DRD2 like), and dopamine transporter (DAT1) in peripheral blood lymphocytes (PBLs) from Lesch–Nyhan patients. We also examined the influence of hypoxanthine in these expressions. As compared to normal PBLs, both ADORA2A and DRD5 expression were abnormal in PBLs from Lesch–Nyhan patients. In contrast, DAT1 expression was similar to control values in HPRT deficient PBLs. These results indicate an abnormal adenosine and dopamine receptor expression in HPRT-deficient cells and suggest disrupted adenosine and dopamine neurotransmission may have a significant role in the pathogenesis of the neurological manifestations of Lesch–Nyhan syndrome.

Variations in c‐Myc and p21WAF1 expression protect normal peripheral blood lymphocytes against BimEL‐mediated cell death

We have examined the effect of suberoylanilide hydroxamic acid (SAHA, Vorinostat, Zolinza) on the viability of normal peripheral blood lymphocytes (PBLs) in vitro and on the expression of 20 apoptosis-related genes. RT-PCR, western blots and flow cytometry were performed to reveal the proteins of apoptosis machinery that were affected to cause cell death. Our data suggest that PBL markedly resisted for approximately 24 h the destructive activity of the agent, but eventually 60% of cells treated with 4 micromol/L SAHA died within 72 h through mitochondrial way of apoptosis. While the expression of the majority of genes remained indifferent against 4 micromol/L SAHA, the cellular levels of BimEL, Bmf-2, Bcl-w and survivin mRNA varied, confirming the pro-apoptotic response of SAHA treated PBL. In addition, the expression of multifunctional proteins c-Myc and p21(WAF1) changed profoundly with the time of SAHA treatment. The Bax activator BimEL increased rapidly, driving cells towards apoptosis likely controlled by c-Myc and p21(WAF1) activities. We suggest that variations in c-Myc and p21(WAF1) expression decelerate the apoptosis in the early period and increase the resistance of resting PBL against SAHA.

Roxithromycin inhibits chemokine-induced chemotaxis of Th1 and Th2 cells but regulatory T cells

Roxithromycin (RXM), a 14-member macrolide antibiotic, has a variety of bioregulatory functions such as anti-inflammatory effects, anti-oxidant effects, and modulation of immune responses. In this study, we analyzed the effect of RXM on chemokine-induced chemotaxis of Th1, Th2, and regulatory T (Treg) cells established from three normal human peripheral blood lymphocytes by the reported methods. Incubation with 10 microM RXM for 18 h did not alter the expression profile of CXCR3 on Th1 cells and CCR4 on Th2 and Treg cells. However, upon RXM preincubation, the migration of Th1 cells to IP-10 and Th2 cells to TARC was partially suppressed, although RXM did not influence Treg cell migration. Erythromycin and clarithromycin at the same concentration did not exert such effects. F-actin polymerization and Ca(++) influx induced by IP-10 and TARC in Th1 and Th2 cells, respectively, was down-regulated by RXM pretreatment. These results imply that RXM exhibits bioregulatory function by influencing chemotaxis of Th1 and Th2 cells while leaving Treg cell migration unaffected.

Small molecule XIAP inhibitors cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells and overcome Bcl-2–mediated resistance

AbstractDefects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. Here, we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells, whereas they do not affect viability of normal peripheral blood lymphocytes, suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential, and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note, XIAP inhibitors even overcome Bcl-2–mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly, XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo, they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus, XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL.

Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene

Background: Loss of genomic imprinting (LOI) of the insulin-like growth factor-2 gene (IGF2) is an epigenetic change involving abnormal activation of the normally silent maternally inherited allele. LOI of IGF-2 gene is found in tumor tissue, normal adjoining mucosa and peripheral blood lymphocytes (PBL) of some patients with colorectal cancer (CRC), suggesting that this alteration precedes and is a risk factor for CRC. However, whether LOI of IGF2 is transitory or remains a permanent epigenetic alteration is unknown. Methods: Standard RT-PCR assays for imprinting analysis of IGF2 were performed on PBL from ApaI informative individuals recruited at baseline and repeated 1 to 3 years later. Prevalence of LOI of IGF2 was also evaluated according to age strata. Results: Four-hundred patients, mean age 60.7 years (range 15-95), 287 (80%) Caucasian were studied. This included 210 (51.4%) patients with no colorectal neoplasia, and 190 (48.6) with colorectal neoplasia. LOI of IGF2 was present in all age strata examined, and no statistically significant association across age strata (p trend > 0.05) was noted. Forty-nine patients had repeat analysis of blood imprinting status at a mean follow up time of 38.2 ± 12.9 months. All but three patients had the same imprinting status at follow up (94% agreement, kappa 0.79, p < 0.001). Genomic imprinting was stable for patients with and without CRN. Conclusion: LOI of the IGF2 gene in PBL appears to be a stable epigenetic phenomenon in most patients. Furthermore, LOI of IGF2 was not associated with age, suggesting an inherited or congenital epigenetic event. These findings support the concept that LOI of IGF2 may be a useful risk factor for CRC predisposition.

GATA-3 Requires C-Myb to Auto-Regulate Its Own Expression in Normal Human Peripheral Blood Lymphocytes Undergoing T Helper Type 2 (Th2) Cell Development

The c-myb protooncogene encodes a transcription factor, c-Myb, which is highly expressed in immature hematopoietic cells. c-Myb is required for many critical aspects of blood cell development including lineage fate selection, proliferation, and at multiple time points during early myeloid, and B and T lymphoid cell development. GATA-3, which belongs to a family of zinc finger transcription factors, is also required at several steps in early T cell development, and specifically in regard to this communication, for the development of T helper type 2 (Th2) cells. A recent study by Maurice et al (EMBO 2007, 26:3629–3640) reported that c-myb regulates T helper cell lineage commitment in developing mouse thymocytes via regulation of GATA-3 expression. As we were unaware of any studies that have addressed the role of c-Myb and GATA-3 in normal human peripheral blood lymphocytes (PBL), we explored the potential regulatory relationship between these transcription factors in cells of this type. Proceeding from the murine studies, we performed a chromatin immunoprecipitation assay (ChIP) which showed that c-Myb bound the GATA-3 downstream promoter in naïve CD4+ T cells under conditions designed to promote Th2 growth. Such binding was not observed in cells stimulated under Th1 promoting conditions. The interaction of c-Myb and GATA-3 proteins was also detected in cell lysates under Th2 cell promoting conditions by immunoprecipitation with both anti-c-Myb, and anti-GATA-3 polyclonal antibodies. Of note, immunoprecipitation with these same antibodies did not show binding of either protein to STAT6. Additional studies revealed that c-Myb activated a GATA-3 minimal promoter by direct binding to a conserved c-Myb binding site in peripheral blood T cells. Of even greater interest, in 293T cells, GATA-3 activated its own promoter ~6 fold when c-Myb was co-expressed in 293T cells. In the absence of c-Myb, GATA-3 did not significantly activate its own promoter in these cells. We have recently shown that c-Myb binds to MLL via menin. A ChIP assay also showed that MLL and Menin bound to the GATA-3 promoter suggesting that c-Myb and GATA-3 form a co-activator complex on the GATA-3 promoter with MLL. Finally, to explore the role of c-myb expression in human peripheral blood naive CD4+ T cells, we employed c-Myb targeted, and control, short hairpin RNA (shRNA) expressed from a lentivirus vector. This strategy yielded a sequence specific 80–90% knockdown of c-Myb expression in our hands. Stimulation of naive peripheral blood CD4+ T cells expressing the c-Myb directed shRNA with cytokines promoting Th2 cell formation (IL-4, IL-2, and anti-IL-12 antibody) blocked the up-regulation of GATA-3 mRNA expression ~90% compared to cells in which a control shRNA had been expressed. Flow cytometric analysis revealed that intracellular IL-4 expression also was diminished. In contrast, silencing c-myb had no effect on T-bet mRNA expression, or intracellular interferon-expression in the cells induced to undergo Th1 cell formation with IL-12, IL-2 and anti-IL-4 antibody. We conclude from these studies that c-Myb regulates developmental programs specific for Th2, as opposed to Th1, cell development. We hypothesize that such control is exerted in peripheral blood T lymphocytes, at least in part, through direct control of GATA-3, whose expression is auto-regulated with the assistance of c-Myb, and perhaps MLL, acting as transcriptional co-factors.

Evaluation of Combinational Therapy of MDM2-Antagonist Nutlin-3 and HDAC-Inhibitor Valproic Acid in Acute Myeloid Leukemia in Vitro and in Vivo

Nutlin-3 is a small-molecule antagonist of MDM2 that induces non-genotoxic stabilization and activation of the tumor suppressor protein p53, resulting in therapeutic effects in tumor models comprising wild type TP53. Valproic acid (VPA) is an anti-convulsive drug with histone deacetylase (HDAC) inhibitor activity that induces differentiation and apoptosis in acute myeloid leukemia (AML) cells. Both therapeutic compounds indirectly affect the regulation of p53; nutlin-3 by inhibiting MDM2, the main negative regulator of p53, and VPA by inhibiting HDACs that participate in p53 deacetylation and destabilization. While mutations in TP53 occur in less than 10% of AML, over-expression of MDM2 is frequently observed. Furthermore, aberrant recruitment of histone deacetylases (HDACs) is seen in AML, leading to block of myeloid differentiation. We therefore hypothesized that concomitant inhibition of MDM2 and HDACs would synergistically induce p53-mediated apoptosis and inhibit tumor growth. We examined the anti-leukemic effects of nutlin-3 in combination with low doses valproic acid in AML cell lines, primary AML cells, and in an in vivo xenograft model using optical imaging. To evaluate the efficacy of the combination of nutlin-3 and VPA in AML cells expressing wild type TP53, the AML cell line MOLM-13 was treated with various concentrations of the two drugs both alone and in combinations (nutlin-3; 0.5–10 μM, VPA; 50–1000 μM, nutlin-3:VPA; 1:100) over different time periods (24–72h, nutlin-3 only for the 24 last hours). Synergistic or additive effects were detected in three distinct viability assays; 3H-thymidine incorporation was used to examine effect on proliferation, WST-1 was used to determine number of metabolic active cells in culture, and DNA specific staining with Hoechst 33342 was used to determine apoptosis after drug-treatment. In addition, MOLM-13 cells treated with the combination showed super additive induction of p53 and target genes. The optimal combination and time period found in MOLM-13 cells were tested in 40 different primary AML samples using the three different viability assays. Toxicity of the combination treatment was tested in normal peripheral blood lymphocytes, while preliminary toxicity of nutlin-3 and VPA alone and in combination on healthy NOD/SCID IL2γnull mice permitted determination of treatment regime. We developed an optical imagable model for in vivo evaluation of the combinational therapy by injecting NOD/SCID IL2γnull mice with MOLM-13 cells transfected with a tetracycline activated luciferase expressing construct (termed L192). Bioluminescent imaging was performed using a TD-SAMI (Time-domain small animal molecular imager). The efficacy of the combinational therapy was tested in 20 NOD/SCID IL2γnull mice injected with MOLM-13 L192 cells and divided into four groups; control, VPA (50 mg/kg b.i.d), nutlin-3 (200 mg/kg b.i.d) and nutlin-3 + VPA (200 mg/kg + 50 mg/kg) b.i.d. Treatment was scheduled for three weeks. The combination of nutlin-3 and VPA significantly inhibited disease development after one week, as monitored by in vivo imaging. Limitations due to nutlin related toxicity prevented further evaluation of continuous combinational therapy after 14 days. However, all treatment groups showed a significant increase in survival compared to the control group, with the combination group demonstrating decreased leukaemic burden as visualized by optical imaging and longer mean average survival time. VPA effect on survival was also tested in a BNML rat leukemia model, in which VPA-treatment (170 mg/kg) resulted in significant longer mean survival compared to the control. Together, the results suggest combined targeting of MDM2 and HDACs as a promising therapeutic approach in AML. Future studies will apply the established bioluminescent MOLM-13 AML xenograft model for further evaluation of the combinational therapy, using a different dosing regimen and scheduling. In addition, we will evaluate combinations of differing classes of HDAC inhibitors and MDM2 antagonists.

Growth inhibitory activity of a novel lectin from Cliona varians against K562 human erythroleukemia cells

In this study, the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians was studied in different cancer cell lines. CvL cytotoxicity was evaluated in mammalian tumor cells and in normal human peripheral blood lymphocytes by the MTT assay using the same range of concentrations (1-150 microg ml(-1)). The mechanisms involved in K562 cell death were investigated by confocal fluorescence microscopy, flow cytometry and immunoblot. CvL inhibited the growth of human leukemia cells, with IC(50) values of 70 and 100 microg ml(-1) for K562 and JURKAT cells, respectively, but it was ineffective on blood lymphocytes and solid tumor cell lines. K562 cell death occurred 72 h after exposure to the lectin and with signs of apoptosis, as analyzed by DAPI and annexin V/PI staining. Investigation of the possible mediators of this process showed that cell death occurred via a caspase-independent pathway. Confocal fluorescence microscopy indicated a pivotal role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished CvL cytotoxic effect. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFkappaB) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and reduced expression of pRb, suggesting that CvL can induce cell cycle arrest. Collectively, these findings indicate an antileukemic effect for CvL and suggest that cathepsin B acts as a death mediator in CvL-induced cytotoxicity possibly in an uncharacterized connection with the membrane death receptor pathway.

Synthesis and recovery of high bioactive phenolics from table-olive brine process wastewater

The generated wastewater of table-olive brine processing contains a high amount of polyphenols which are endowed with interesting biological activities. The ethyl acetate extract (EAE) of such wastewater shows high hydroxytyrosol (HT) and tyrosol concentrations of 690 and 98 mg g −1 dry weight extract, respectively. Phenolic compounds analysis was performed by gas chromatography–mass spectrometry (GC–MS). The antioxidant activity was evaluated by the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonate] radicals. Total polyphenol content was estimated with the Folin–Ciocalteu assay. Hydroxytyrosol has DPPH and ABTS radicals scavenging activities higher than 2,6-di- tert-butyl-hydroxytoluene (BHT), while triacetylated hydroxytyrosol (triAcHT) was devoid of any antioxidant activity. The assessment of these antioxidant compounds in biological systems was carried out by the determination of their in vitro cytotoxicity against two different human cancer cell lines (HeLa and DG75) and normal peripheral blood lymphocytes (PBL) using the MTT assay. The 50% cytotoxic concentrations were ranged between 27 and 210 μg mL −1 for Hela and DG75. At the same concentration range, the EAE and the pure HT and triAcHT exhibited an insignificant cytotoxicity against PBL. Incubation of HeLa and DG75 cells with non-cytotoxic concentrations of EAE, HT or triAcHT resulted in a remarkable protection from the oxidative stress induced by Fe 2+. The antimicrobial activity evaluated by the broth dilution NCCL method using Gram-positive ( Staphylococcus aureus, Bacillus subtilis) and Gram-negative ( Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica) bacteria and ( Candida albicans, Aspergillus niger) fungi showed a broad spectrum bactericidal and fungicidal effect of table-olives-EAE, HT and triAcHT. The MICs vary from 125 to 500 μg mL −1 for bacteria and from 500 to 2000 μg mL −1 for fungi.

Apogossypolone, a nonpeptidic small molecule inhibitor targeting Bcl-2 family proteins, effectively inhibits growth of diffuse large cell lymphoma cells in vitro and in vivo

Apogossypolone (ApoG2) is a semi-synthesized derivative of gossypol. The principal objective of this study was to compare stability and toxicity between ApoG2 and gossypol, and to evaluate anti-lymphoma activity of ApoG2 in vitro and in vivo. ApoG2 shows better stability when compared with a racemic gossypol and can be better tolerated by mice compared to gossypol. ApoG2 showed significant inhibition of cell proliferation of WSU-DLCL2 and primary cells obtained from lymphoma patients, whereas it displayed no toxicity on normal peripheral blood lymphocytes. For a treatment of 72 h, the IC50 of ApoG2 was determined to be 350 nM against WSU-DLCL2 cells. Treatment with ApoG2 at 600 mg/kg resulted in significant growth inhibition of WSU-DLCL2 xenografts. When combined with CHOP, ApoG2 displayed even more complete inhibition of tumor growth. ApoG2 binds to purified recombinant Bcl-2, Mcl-1, and Bcl-XL proteins with high affinity and is shown to block the formation of heterodimers between Bcl-XL and Bim. For a treatment of 72 h, ApoG2 induced a maximum of 32% of apoptotic cell death. Western blot experiments showed that treatment with ApoG2 led to cleavage of caspase-3, caspase-9 and PARP. Moreover, pretreatment of DLCL2 cells with caspase-3, -9 and broad spectrum caspase inhibitors significantly blocked growth inhibition induced by ApoG2. In conclusion, ApoG2 effectively inhibits growth of DLCL2 cells at least partly by inducing apoptosis. It is an attractive small molecule inhibitor of the Bcl-2 family proteins to be developed further for the treatment of diffuse large cell lymphoma.

The inductive effect of interleukin-4 on IgG4 and IgE synthesis in human peripheral blood lymphocytes

Using murine monoclonal antibodies against human IgG subclasses, specific and sensitive ELISAs assay to quantify the four human IgG subclasses in cell culture supernatants were established. The effect of human recombinant interleukin-4 (IL-4) on the regulation of IgG subclasses by normal peripheral blood lymphocytes was investigated. In addition to the enhancement of IgE synthesis, IL-4 preferentially induced IgG4 synthesis in vitro, whereas IL-4 had no effect on IgG1, IgG2, and IgG3 synthesis. IL-4-induced IgG4 production was blocked in a dose-dependent manner by recombinant interferon-gamma and anti-human IL-4 monoclonal antibody. Collectively, this data indicates that IL-4 plays an important regulatory role in both IgG subclass and IgE synthesis.

The human T cell receptor V beta repertoire of normal peripheral blood lymphocytes before and after mitogen stimulation.

Mitogen stimulation of T cells in vitro has been employed in the analysis of the T cell antigen receptor (TCR) repertoire and as a method of generating T cell lines and clones. It has been suspected for some time that mitogen stimulation may bias the repertoire. We have addressed this problem employing a semi-quantitative technique utilizing the polymerase chain reaction (PCR) and flow cytometry. Using this PCR method and a panel of primers to 22 V beta subgroups, the V beta repertoire of both unstimulated and phytohaemagglutinin (PHA)-stimulated peripheral T cells from eight healthy individuals was investigated. The samples were also analysed by flow cytometry using anti-V beta 2, V beta 5 and V beta 8 MoAbs. A significant increase in the expression of V beta 6, V beta 7.2 and V beta 10.1 was found in all eight samples of PHA-stimulated T cells compared with unstimulated T cells using the PCR method. In contrast, no differences were found between unstimulated and PHA-stimulated T cells by flow cytometry. These results question the validity of using mitogen-stimulated T cells to investigate TCR gene usage.

Individual germinal centres of myasthenia gravis human thymuses contain polyclonal activated B cells that express all the VH and VK families

Using in situ hybridization, we analysed the immunoglobulin repertoire expressed by the B cells present in myasthenia gravis thymuses from four patients. B cells, mostly in activated state, were clustered in germinal centres, in which multiple isotypes were identified. A majority of cells expressed IgG as compared with IgM, with a roughly similar contribution of kappa and lambda chains. Hybridization with the six VH and the 4 VK human family probes was observed in serial sections, providing additional evidence that individual germinal centres were polyclonal. The thymic B cell repertoire closely reflected the VH and the VK family usage of normal peripheral blood lymphocytes with the preferential utilization of VH3, VK1 and VK3.

A study on the genotoxic effects of 8-Cl-cAMP on human lymphocytes in vitro

8-chloro-cyclic adenosine 3',5'-monophosphate (8-Cl-cAMP) is the most potent cAMP analogue that selectively inhibits a variety of cancer cell lines in vitro and tumors in vivo. Its action toward a variety of tumors, especially when coupled with other antitumor agents, have lead to phase I clinical investigations and recently phase II clinical investigations. Until today very little was done to evaluate its genotoxic potential. In order to evaluate its genotoxic potential we used the cytogenetic and cytokinesis block micronucleus assay in vitro on peripheral blood lymphocytes of healthy individuals. Using three concentrations (1 microM, 5 microM and 15 microM), 8-Cl-cAMP in normal human peripheral blood lymphocytes did not induce any cytogenetic aberrations of the structural type [chromatid breakage, isochromatid breakage and gaps], but did induce premature centromere separation (PCS) in all respective doses and increased the frequency of micronuclei (p <0.05) only in the highest dose (15 microM). Antiproliferative action of 8-Cl-cAMP was estimated by using the cytokinesis block nuclear division index (NDI). The results showed a decrease in the NDI of cells exposed to all doses of 8-Cl-cAMP when compared to control. Therefore, the overall results show a genotoxic potential of 8-Cl-cAMP in peripheral blood lymphocytes in vitro.

490 Loss of IGF2 Genomic Imprinting and Cancer Risk in An Ethnically Diverse Population

[Background] Loss of genomic imprinting (LOI) of IGF-2 is an independent risk factor for colorectal cancer. However, whether LOI of IGF-2 is a risk factor for other malignancies remains unclear. [Aim] The aim of this study was to determine if LOI of IGF-2 was associated to prevalent cases of breast, prostate, colorectal cancer (CRC)and other non-colorectal cancers. [Patients and Methods] Consecutive patients who underwent colonoscopic examination at three Academic Centers were recruited. We performed PCR on gDNA and RT-PCR on RNA extracted from normal peripheral blood lymphocytes (PBL) of prospectively recruited patients, followed by ApaI digestion. Based on ApaI DNA polymorphism, the informative (heterozygosity) and imprinting status of IGF2 gene were determined. Statistical analysis was performed using multiple logistic regression models, Odds Ratios (OR) and 95% CI were calculated with STATA 9.0. [Results] A total of 367 informative patients (based on ApaI DNA polymorphism) were recruited; mean age 56.4 (range 21-85); 166(45%) female; and Hispanic 73(20%). Based on baseline colonoscopy and review of medical records, there were 164 (44.7%) controls and 128 prevalent cancer patients including: colon (112), rectal (33), breast (15), prostate (15), other non-CRC (22). LOI-positive individuals had statistically significant OR for colon, rectal, breast, non-colorectal cancers (See Table below). LOI-positive patients had a borderline increased risk for prostate cancer (OR = 1.73, 95% CI 0.88-6.31) compared to LOI-negative patients. [Conclusion] LOI-positive individuals had a statistically significant 2-3 fold increased risk of having colon, rectal, breast and non-CRC cancers compared to LOI-negative individuals. LOI of IGF2 might be a biomarker for cancer risk, and perhaps may be in the carcinogenic pathway of multiple epithelial neoplasias. [Support] NIH grants K22 CA-, U54 CA-096297, RCMI and P50 CA-62924-10, The Doris Duke Charitable Foundation, The John G. Rangos Sr. Charitable Foundation, and The Clayton Fund. Loss of Imprinting and Cancer Risk

BimEL‐dependent apoptosis induced in peripheral blood lymphocytes with n‐butyric acid is moderated by variation in expression of c‐myc and p21(WAF1)

We have examined the effect of sodium butyrate (SB) on the viability of normal peripheral blood lymphocytes (PBLs) in vitro and the effect of this agent on the expression of 20 apoptosis-related genes. Data suggest that PBL treated with 2 mmol L(-1) SB resisted for at least 8 h the destructive activity of the agent, but eventually 30% of cells died within 72 h. As documented by flow cytometry and cytochrome c release study, cells underwent mitochondrial-derived apoptosis. While the expression of the majority of genes examined by RT-PCR and Western blots remained indifferent to 2 mmol L(-1) SB, the cellular levels of BimEL, c-myc, p53, and p21(WAF1) varied profoundly with the time of SB treatment. The Bax activator BimEL increased rapidly, driving cells toward apoptosis likely controlled by c-myc and p21(WAF1) activities. The c-myc, exercising the role of mediator of the function of BimEL and inhibitor of p21(WAF1) expression, decreased significantly for several hours after adding SB but within 48 h it returned to close to its original value. An apoptosis inhibitor and executive caspase substrate p21(WAF1) increased early at the beginning of treatment but subsequently, within a time frame of 72 h, profoundly dropped in terms of both a caspase-dependent and caspase-independent way. We suggest that variations in c-myc and p21(WAF1) expression delay apoptosis making PBL resistant to SB for several hours, and together with fast catabolism of SB in vivo protect PBL against the destructive activity of this anti-cancerous metabolite of colonic bacteria.

Inhibitory effect of wheat bran feruloyl oligosaccharides on oxidative DNA damage in human lymphocytes

The present work assessed the protective effect of feruloyl oligosaccharides (FOs), the ferulic acid ester of oligosaccharides from wheat bran, against oxidative DNA damage in normal human peripheral blood lymphocytes induced by hydrogen peroxide (H2O2). The DNA damage was measured by using the single cell gel electrophoresis assay (comet assay). Lymphocytes were subjected to DNA damage by exposure to a range of H2O2 concentrations (10–200μmol/l). H2O2, at a concentration of 200μmol/l, resulted in nearly all cells being highly damaged. FOs showed no cytotoxicity and genotoxicity to normal human lymphocytes at the tested concentrations (10–500μmol/l). In addition, DNA damage in human lymphocytes induced by 100μmol/l H2O2 was inhibited by FOs in a concentration-dependent fashion with 91.1% inhibition of lymphocyte DNA damage at 500μmol/l as compared with the control. The results suggest that water-soluble FOs from wheat bran are able to enhance the ability of human lymphocytes to resist H2O2 induced oxidative damage.

Protein Expression of p15 and p21 Plays an Unfavorable Prognostic Role in Adult Acute Lymphoblastic Leukemia (ALL) Patients Independently of Their Gene Promoter Methylation Status.

Epigenetic silencing of tumor suppressor (TS) genes is a hallmark in human leukemias, particularly through DNA methylation. Cyclin-dependent kinase inhibitors (CKI) are, among other genes, frequently found methylated in their promoter region. This epigenetic modification has been described also in acute lymphoblastic leukemia (ALL). However, the relationship between aberrant DNA methylation and protein expression of TS genes has not yet been extensively evaluated in adult ALL series. The aim of this study was to analyze in primary cells from newly diagnosed adult ALL patients, uniformly treated according to the LAL2000 GIMEMA protocol, the promoter methylation status of p73, p21, p15 and p16, evaluating in addition the p21, p15 and p16 protein expression. The DNA methylation status of promoter regions was investigated, according to cell availability, using a widely accepted method based on bisulfite modification of DNA, followed by methylation-specific PCR assay (MSP). Protein expression was evaluated by Western blot. Normal peripheral blood lymphocytes, as already described, resulted unmethylated for p73, p21, p15 and p16, and did not express the p21, p15 and p16 proteins. In ALL patients, in contrast, only the p21 promoter region was found constantly unmethylated. The p15, p16 and p73 promoter genes were found methylated in 15/37 (40.5%), 8/43 (18.6%) and 9/36 (25%) patients, respectively. Only 2/23 cases (8.6%) resulted simultaneously methylated for p15, p16 and p73. The p21 and p15 protein expression was found in 28/85 (32.9%) and 44/85 cases (51.8%), respectively. The p16 protein, in contrast, was never expressed. The p16 methylation was associated with the T-ALL (P=0.005) phenotype and with higher white blood cell (WBC) counts (P=0.027). Resistance to spontaneous induction of apoptosis was significantly associated with p21 protein expression (P=0.019) and its co-expression with p15 (P=0.049). Achievement of CR was not influenced by gene methylation status, nor by single protein expression. Interestingly, the co-expression of p15 and p21 was associated with failure to induction treatment: only 6/63 (9.5%) patients co-expressing p15 and p21 obtained a CR (P=0.027). Multivariate analysis confirmed the unfavorable role of this protein co-expression (P=0.059) on CR achievement. In contrast, once patients achieved remission, p21 protein expression was associated with a prolonged DFS, as confirmed by multivariate analysis for DFS (P=0.039). In conclusion, p15 and p21 protein expression plays an unfavorable prognostic role in adult ALL patients independently of the p73, p21, p15 and p16 gene promoter methylation status.

C-Myb Contributes to Normal Human Peripheral Blood T Helper 2 Cell Development through Regulation of GATA-3 Expression.

c-Myb is an obligate hematopoietic transcription factor which is highly expressed in immature hematopoietic cells. It plays a critical role in both myeloid and lymphoid cell development, and specifically in regard to this communication, at multiple points during early T cell development. While the role of c-Myb in developing cells has been intensively studied, we noted that there is a relative paucity of investigations focused on c-myb function in peripheral blood T cells. This situation exists despite the relatively high level of c-myb expression we observe in unstimulated cells, and the increase that occurs when such cells are stimulated. Very recently (Embo J, Aug 2007), Maurice et al demonstrated that c-myb regulates T helper cell lineage commitment in developing mouse thymocytes, at the same time that it appears to block development of cytotoxic T cells, via regulation of GATA-3. However, the role of c-Myb and GATA-3 in normal human peripheral blood lymphocytes was not explored. Here we show that c-myb regulates GATA-3 expression directly in peripheral blood CD4+ cells and has a critical role in human Th2 cell development. To explore the role of c-myb expression in human peripheral blood naive CD4+ cells we employed c-Myb targeted, and control, short hairpinRNA (shRNA) expressed from a lentivirus vector. This strategy yielded a sequence specific ~ 80–90% knockdown of c-Myb expression. Stimulation of naive peripheral blood CD4+ T cells in which the c-Myb directed shRNA was expressed, with a cocktail designed to promote Th2 cell formation (IL-4, IL-2, and anti-IL-12 antibody) blocked the up-regulation of GATA-3 mRNA expression ~90% compared to cells in which a control shRNA had been expressed. Flow cytometric analysis showed that intracellular interleukin-4 expression was also diminished in CD4+ cells stimulated under Th2 promoting conditions. In contrast, silencing c-myb did not affect T-beta mRNA expression, or intracellular interferon-γ expression in the cells induced to undergo Th1 cell formation with IL-12, IL-2 and anti-IL-4 antibody. A ChIP assay showed that c-myb bound to the GATA-3 promoter in human primary CD4+ cells stimulated under Th2 cell promoting conditions, but not under Th1 promoting conditions. A reporter assay demonstrated that c-myb over-expression increased GATA-3 promoter activity by ~5 fold in 293T cells, and approximately 3 fold in human primary T cells. Silencing c-myb in primary human T cells with shRNA resulted in an approximately 50% decrease in GATA-3 promoter activity. These results demonstrate that c-myb plays an important role in Th2 cell development at least in part through direct regulation of GATA-3 expression. In primary human effector/memory CD4+ T cells, which includes established Th2 cells, c-myb suppression with shRNA also decreased GATA-3 promoter activity by approximately 85%, but the suppression of IL-4 expression was only moderate (~50%). These results suggest that c-myb may also play a role in the homeostasis of established Th2 cells. Finally, and as might be expected, silencing c-myb suppressed proliferation of naive CD4+ cells. We conclude that c-Myb plays multiple roles in human peripheral blood T lymphocytes, including the generation and maintainence of Th2 cells, in addition to regulation of cell proliferation. It performs these functions, at least in part, through direct regulation of GATA-3.

Enhancement of lymphocyte migration and cytokine production by ephrinB1 system in rheumatoid arthritis

Although the etiology of early events in rheumatoid arthritis (RA) remains undefined, an anomaly in T cell homeostasis and hyperproliferation of synovial-lining cells are involved in the disease process. Since it has been reported that the ephrin/Eph receptor system plays important signaling roles in inflammation processes, we attempted to examine ephrinB molecules in T cells and synovial cells derived from RA in this study. The expression level of ephrinB1 was significantly high in synovial fibroblasts and CD3-positive exudate lymphocytes in synovial tissues derived from patients with RA compared with those in osteoarthritis (OA). Protein and mRNA levels of ephrinB1 were also higher in peripheral blood lymphocytes (PBLs) prepared from patients with RA than those from normal controls. Similar results were obtained from an animal model of human RA, collagen antibody-induced arthritis mice. Moreover, a recombinant ephrinB1/Fc fusion protein stimulated normal PBLs to exhibit enhanced migration and production of TNF-alpha. EphrinB1/Fc also activated synovial cells established from patients with RA to produce IL-6. Tyrosine phosphorylation of EphB1 was induced in these cells by ephrinB1/Fc. The CpG islands in the 5' upstream regulatory region of the ephrinB1 gene were hypomethylated in RA patients compared with those of normal donors. These results suggest that ephrinB1 and EphB1 receptors play an important role in the inflammatory states of RA, especially by affecting the population and function of T cells. Inhibition of the ephrinB/EphB system might be a novel target for the treatment of RA.