Hematopoietic Tumor Cells Research Articles

Mda-7/IL-24 inhibits the proliferation of hematopoietic malignancies in vitro and in vivo

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) has been consistently shown to exert growth inhibitory effects on various tumor types. However, the majority of these reports were limited to solid tumors. The purpose of this study was to investigate the antitumor activity of mda-7/IL-24 and the underlying mechanism in hematopoietic malignancies. We determined the expression of mda-7/IL24 and its heterodimeric receptors in hematopoietic tumor cell lines and then stably transfected mda-7/IL-24 into K562 (leukemia) and Namalwa (lymphoma) cell lines to assess the effects of mda-7/IL-24 on cell proliferation, cell cycle, apoptosis, colony-forming ability, and tumor growth in vivo. Microarray analysis was performed to determine the genes that were differentially regulated by mda-7/IL-24 in K562 cells. Expression of mda-7/IL-24 or its intact receptor pairs was not detected in the 11 cell lines tested. Ectopic expression of mda-7/IL-24 induced significant (p < 0.05) inhibition of cell growth and colony formation in both K562 and Namalwa cells, and the growth inhibition in K562 cells was associated with G(0)/G(1) cell-cycle arrest. Results of in vivo studies showed good correlation with in vitro inhibition of tumor cell proliferation in both the cell lines. We also showed that the increase in p21(WAF-1) and BCCIP and decrease in cdk6, smurf2, and phosphorylated pRb, which are regulators of cell-cycle progression, might account for G(0)/G(1) cell-cycle arrest in K562 cells. The present study demonstrated for the first time the potential antitumor activity of mda-7/IL-24 in chronic myelogenous leukemia and lymphoma.

GS-9219—A Novel Acyclic Nucleotide Analogue with Potent Antineoplastic Activity in Dogs with Spontaneous Non–Hodgkin's Lymphoma

GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl)guanine (PMEG), was designed as a cytotoxic agent that preferentially targets lymphoid cells. Our objective was to characterize the antiproliferative activity, pharmacokinetics, pharmacodynamics, and safety of GS-9219. GS-9219 was selected through screening in proliferation assays and through pharmacokinetic screening. The activation pathway of GS-9219 was characterized in lymphocytes, and its cytotoxic activity was evaluated against a panel of hematopoietic and nonhematopoietic cell types. To test whether the prodrug moieties present in GS-9219 confer an advantage over PMEG in vivo, the pharmacokinetics, pharmacodynamics (lymph node germinal center depletion), and toxicity of equimolar doses of GS-9219 and PMEG were evaluated after i.v. administration to normal beagle dogs. Finally, proof of concept of the antitumor efficacy of GS-9219 was evaluated in five pet dogs with spontaneous, advanced-stage non-Hodgkin's lymphoma (NHL) following a single i.v. administration of GS-9219 as monotherapy. In lymphocytes, GS-9219 is converted to its active metabolite, PMEG diphosphate, via enzymatic hydrolysis, deamination, and phosphorylation. GS-9219 has substantial antiproliferative activity against activated lymphocytes and hematopoietic tumor cell lines. In contrast, resting lymphocytes and solid tumor lines were less sensitive to GS-9219. GS-9219, but not PMEG, depleted the germinal centers in lymphoid tissues of normal beagle dogs at doses that were tolerated. In addition, GS-9219 displayed significant in vivo efficacy in five dogs with spontaneous NHL after a single administration, with either no or low-grade adverse events. GS-9219 may have utility for the treatment of NHL.

Synthesis of antiproliferative Cephalotaxus esters and their evaluation against several human hematopoietic and solid tumor cell lines: uncovering differential susceptibilities to multidrug resistance.

Deoxyharringtonine (2), homoharringtonine (3), homodeoxyharringtonine (4), and anhydroharringtonine (5) are reported to be among the most potent members of the antileukemia alkaloids isolated from the Cephalotaxus genus. Convergent syntheses of these four natural products are described, each involving novel synthetic methods and strategies. These syntheses enabled evaluation of several advanced natural and non-natural compounds against an array of human hematopoietic and solid tumor cells. Potent cytotoxicity was observed in several cell lines previously not challenged with these alkaloids. Variations in the structure of the ester chain within this family of alkaloids confer differing activity profiles against vincristine-resistant HL-60/RV+, signalling new avenues for molecular design of these natural products to combat multi-drug resistance.

Protein expression of BLM gene and its apoptosis sensitivity in hematopoietic tumor cell strains

Patients with Bloom syndrome (BS) show an immunodeficiency, an enhanced sister chromatid exchanges (SCEs), a strong genetic instability and an increased predisposition to all. In order to investigate the differential expression of BLM protein in hematopoietic tumor cell strains and study the effects of BLM gene on ultraviolet (UV)-or hydroxyurea (HU)-induced apoptosis, Western blot was used to detect the expression of BLM protein in normal human bone marrow mononuclear cells and 4 kinds of hematopoietic tumor cell strains. The 4 kinds of hematopoietic tumor cells were exposed to UV light with a germicidal UV lamp or treated with 2 mmol/L hydroxyurea and the apoptotic rate was detected by using AnnexinV-FITC. The results showed that these tumor cells expressed BLM protein higher than the normal human bone marrow mononuclear cells (P<0.01). In the 4 hematopoietic tumor cells, BLM protein was all specially cleaved in response to UV-or HU-induced apoptosis. The increase of BLM protein expression may play an important role in the development of these tumors, and BLM proteolysis is likely to be a general feature of the apoptotic response.

T Cell Therapy in Allogeneic Stem Cell Transplantation

T cells derived from healthy stem cell donors can mediate curative immune responses after allogeneic stem cell transplantation (SCT) by eliminating (residual) hematopoietic tumor cells life-threatening viral infections. The first evidence for the crucial role of T cells came from the observations that depletion of T cells from a stem cell graft to prevent the development of acute graft versus host disease (GVHD) was associated with an increased incidence of relapse of the malignancy and life threatening viral infections including CMV disease and EBV mediated post-transplant lymphoproliferative disease (PTLD). Apparently, after allogeneic SCT T cells are not only responsible for the detrimental GVHD, but also can mediate a graft versus tumor (GVT) effect and are essential for long term of control of persistent endogenous viral infections. Unmanipulated T cell populations derived from the stem cell donor administered to the patient may result in concurrent development of GVHD, GVT and anti-virus responses. Immune suppression to control GVHD may lead to reduction or elimination of GVT and pathogen specific T cell responses. Obviously, if T cell responses leading to GVHD could be separated from GVL or pathogen-specific reactivity, morbidity and survival after allogeneic SCT can be significantly improved. One approach to manipulate the effect of donor T cells after transplantation has been the depletion of donor T cells from the graft with postponed administration of donor T cells 1. The rationale for this approach came from several mouse and human studies illustrating that the recipient microenvironment plays a significant role in the development of GVHD2. Tissue damage during the conditioning regimen leading to the initiation of a ‘cytokine storm’, the strong lymphodepletion after transplant leading to homeostatic proliferation of donor T cells shortly after transplant, and the presence of patient derived activated dendritic cells (DC) which are required for the induction of allo-immune responses all contribute to these strong allo-response immediately after transplantation. Postponed T cell administration at the time of replacement of the majority of recipient DC by donor DCs, after repair of the tissue damage, and after partial reconstitution of T cells in peripheral blood of patients is associated with lower risk of developing severe acute GVHD. Administration of T cells from 6 months after transplantation can be performed with relatively low risk of inducing severe GVHD, although chronic GVHD remains a significant complication. However, recurrence of the malignancy and the development of severe opportunistic infections frequently take place within the first half year after transplantation. Thus, although postponed administration of T cells can result in less severe GVHD, overall survival did not significantly improve. Delivering stem cell transplants that are free from GVHD while conferring enhanced protection against malignant disease recurrence and viral reactivation is considered by many transplanters to be a key goal (the “Holy Grail”) for improving transplant success. We will discuss several approaches for the selective administration of non-GVHD reactive donor T cells: selective allodepletion, adoptive transfer of virus-specific T cells, allo-reactive anti-tumor T cells recognizing minor histocompatibility antigens (mHag) or MHC molecules, or T cells recognizing tumor-associated self antigens.

Induction of apoptosis of myeloid leukemia with CSC17.1, a unique monoclonal antibody targeting CD45RA

3030 Background: Blast crisis from myeloid leukemia is may be responsive to chemotherapy, but a majority of patients die from their disease. Relapse may be due to persistence of cancer stem cells (CSC). A monoclonal antibody (mAb) that targets myeloid blast cells including the CSC without targeting the hematopoietic stem cell (HSC: CD34+90+) may be of value. CSC phenotype of CML-BC (CD34+38+123+45RA+) and AML (CD34+38–90-) have been defined. Method: We developed a biologically based mAb platform with 2 important steps after generating the mAb library: i) mAb must not bind the HSC (SS; safety screen); and ii) must bind the target cells (RxS; therapeutic screen). HSC for the SS were from G-CSF mobilized blood; cells for RxS was patient specimens of CML-BC and AML. After screens, the target was indentified, in vitro and in vivo functional assays are performed. Results: 5 of 400 mAb passed the SS and RxS. CSC17.1, an IgG2b isotype binding a unique, linear, 12 amino acid sequence of CD45RA has been selected. CML (Kasumi4), AML (Kasumi3 and KG1a), ALL (MOLT3) and NHL (Pfeiffer) cel lines express high levels of CD45RA. Five of 5 CML-BC and 3 of 3 AML patient specimens express CD45RA in high levels. Kausmi4 and Pfeiffer cell lines and patient specimens of CML-BC and AML undergo apoptosis in a dose and time dependent manner after exposure to CSC17.1 (&gt;80% apoptosis at 1ug/ml@8h). KG1a is refractory to apoptosis despite high levels of target on the cell surface (15% apoptosis at 1ug/ml@8h). Normal T,B and NK cells express high levels of CD45RA, but do not undergo apoptosis or activation when exposed to CSC17.1. CSC17.1 causes capping and patching, but not internalization of CD45RA in cell lines and CD8+ normal cells. Commercially available antibodies to CD45RA do not inhibit binding of CSC17.1 to CD45RA, do not cause apoptosis, do not cause patching and capping, but do undergo internalization. In vivo murine models with cell lines and patient specimens of CML-BC and AML are underway. Conclusions: 1) CSC17.1 binds a previously undefined epitope of CD45RA that causes apoptosis of selected hematopoietic tumor cell lines and patient specimens of CML-BC and AML; 2) apoptosis, is a unique function of CSC17.1 compared to other mAb against CD45RA; 3) CML-BC and AML CSC include the CD45RA antigen suggesting CSC17.1 will target these cells. No significant financial relationships to disclose.

Inhibition of cell proliferation by lenalidomide is associated with stimulation of Egr1 transcriptional activity in a chromosome 5 deleted Burkitt’s lymphoma and multiple myeloma cell line

8110 Background: The mechanism by which lenalidomide exerts its anti-proliferative effects in deletion 5q MDS clones or MM cells is not yet fully elucidated. Early growth response (Egr1) gene is a tumor suppressor gene located on chromosome 5q31.1 that encodes a transcription factor involved in the regulation of cell proliferation and apoptosis. We hypothesized that lenalidomide may act by enhancing the expression or activity of Egr1 in sensitive hematopoietic tumor cells, especially those with only a single copy of the Egr1 gene. Methods: Transcriptional activity was measured using luciferase reporter plasmids. Gene knockdown and expression studies used siRNA technology and qRT-PCR, respectively. Cell proliferation was measured by 3H-thymidine incorporation. Results: Lenalidomide stimulated the transcriptional activity of Egr1 in the lenalidomide-sensitive chromosome 5 deleted Burkitt's lymphoma Namalwa CSN.70 and in the MM cell line LP-1. Egr1 siRNA Namalwa cells proliferated more than mock controls, indicating that Egr1 functions as a tumor suppressor in Namalwa cells. Lenalidomide had no effect on expression of Egr1, but augmented Egr1 nuclear transport in a dose-dependent manner. Lenalidomide did not affect expression of the Egr1 downstream effector genes ATF3, fibronectin, p53, PTEN, and TGF-β1, while p21 levels increased. However, lenalidomide-induced p21 expression was not affected in Egr1 siRNA Namalwa cells. Interestingly, lenalidomide’s anti-proliferative potency was greater in Egr1 siRNA Namalwa but not in Egr1 siRNA LP-1 cells. Conclusions: Lenalidomide induces nuclear transport and transcriptional activation of the tumor suppressor Egr1, which may contribute to lenalidomide’s anti-proliferative activity in a non-p21 dependent manner. This activity may be related to the levels of Egr1 expression, explaining why del 5q31 myelodysplastic clones are especially sensitive to the cytotoxic effects of lenalidomide. No significant financial relationships to disclose.

Interaction of a Cyclin E Fragment with Ku70 Regulates Bax-Mediated Apoptosis

The cyclin E/Cdk2 complex plays an essential role in the G(1)/S cell cycle transition and DNA replication. Earlier we showed that in hematopoietic tumor cells, caspase-mediated cleavage of cyclin E generates p18-cyclin E, which is unable to interact with Cdk2 and therefore plays a role independent of the cell cycle. The expression of a cleavage-resistant cyclin E mutant greatly diminishes apoptosis, indicating the critical role of cyclin E cleavage. p18-cyclin E expression can induce apoptosis or sensitization to apoptotic stimuli in many cell types. Here we identify Ku70 as a specific p18-cyclin E-interacting partner. In hematopoietic tumor cell lines, the association of p18-cyclin E with Ku70 induces the dissociation of Bax from Ku70, followed by Bax activation. This mechanism of Bax activation leads to the amplification of the apoptosis signal in all tumor cell lines examined. N-terminal Ku70 deletion mutants are unable to bind to p18-cyclin E to regulate its apoptotic effect. p18-cyclin E-mediated amplification of apoptosis is dependent on Bax and Ku70 being greatly diminished in Ku70(-/-) and Bax(-/-) mouse embryo fibroblasts and in hematopoietic cells where Bax knockdown was achieved by short interfering RNA. The p18-cyclin E/Ku70 and Bax/Ku70 interactions provide a balance between apoptosis and the survival of cells exposed to genotoxic stress.

Cytotoxic Evaluation of Four Haplophyllum. Species with Various Tumor Cell Lines

Total extracts of the aerial parts of four Haplophyllum. species (Rutaceae) including Haplophyllum stapfianum. Hand.-Mazz., H. tuberculatum. (Forssk.) Adr. Juss., H. acutifolium. (DC.) G. Don, and H. viridulum. Soják were tested for in vitro. cytotoxicity against 11 tumor cell lines and normal peripheral blood mononuclear cells (PBMCs). Cytotoxicity of Haplophyllum. species were also compared with Ruta graveolens. L. (Rutaceae) extract. Cytotoxic activity of extracts was evaluated by the WST-1 assay and expressed as IC50 values. The results obtained indicated that among hematopoietic tumor cell lines, strong cytotoxic activity was observed from H. stapfianum., H. tuberculatum., and H. acutifolium. against RAMOS cell line with IC50 values of 12.3, 25.3, and 23.7 µg/mL, respectively; H. stapfianum. and H. tuberculatum. against U937 cell line with IC50 values of 15.6 and 29.3 µg/mL; and H. tuberculatum. against RPMI-8866 cell line with IC50 = 31.8 µg/mL. Testing plant extracts on solid tumor cell lines revealed that all Haplophyllum. species have markedly high cytotoxicity against LNCap-FGC-10, a prostate adenocarcinoma cell line, except H. viridulum.. H. tuberculatum. also showed strong cytotoxic effect against 5637, a bladder carcinoma cell line (IC50 = 23.3 µg/mL). Investigating cytotoxic activity of R. graveolens. resulted in higher IC50 values against all tumor cell lines except LNCap-FGC-10 (IC50 = 27.6 µg/mL). Cytotoxicity evaluation of Haplophyllum. species on PBMCs revealed that resulting IC50 values for H. stapfianum. and H. acutifolium. on tumor cell lines with high cytotoxic activity were significantly lower than the IC50 values obtained for PBMCs (p < 0.05).

Skeletal metastasis: Established and emerging roles of parathyroid hormone related protein (PTHrP)

Parathyroid hormone related protein (PTHrP) is a well characterized tumor derived product that also has integral functions in normal development and homeostasis. PTHrP is produced by virtually all tumor types that metastasize to bone and numerous studies have demonstrated a correlation between PTHrP expression and skeletal localization of tumors. PTHrP has prominent effects in bone via its interaction with the PTH-1 receptor on osteoblastic cells. Through indirect means, PTHrP supports osteoclastogenesis by upregulating the receptor activator of NFkappaB ligand (RANKL) in osteoblasts. PTHrP also regulates osteoblast proliferation and differentiation in manners that are temporal and dose dependent. Bone turnover has been implicated in the localization of tumors to bone and PTHrP increases bone turnover. Bone turnover results in the release of growth factors such as TGFbeta and minerals such as calcium, both of which impact tumor cell growth and contribute to continued PTHrP production. PTHrP also has anabolic properties and could be in part responsible for osteoblastic type reactions in prostate cancer. Finally, emerging roles of PTH and PTHrP in the support of hematopoietic stem cell development in the bone marrow microenvironment suggest that an interaction between hematopoietic cells and tumor cells warrants further investigation.

β1 integrin mediated adhesion increases Bim protein degradation and contributes to drug resistance in leukaemia cells

It has been shown that the tumour microenvironment confers resistance to chemotherapy. Specifically, it was previously reported that adhesion of haematopoietic tumour cells to fibronectin (FN) via beta1 integrins confers a multi-drug resistance phenotype commonly referred to as cell adhesion mediated drug resistance. The present study showed that the pro-apoptotic BCL-2 family member Bim was reduced when leukaemia cells were adherent to FN via beta1 integrins. beta1 integrin-mediated regulation of Bim in K562 cells was demonstrated to be partly a result of increased proteasomal-mediated degradation of Bim protein levels, and proteasome inhibitors prevent Bim degradation. Increased degradation of Bim was not related to activation of the mitogen-activated protein kinase pathway, as adhesion of K562 cells caused a reduction in phospho-extracellular signal-related kinase (ERK)1/2 levels. In addition, pharmacological inhibition of MAP/ERK (MEK) with PD98059 did not increase Bim levels. Reducing Bim levels by short hairpin RNA targeting inhibited imatinib and mitoxantrone-induced cell death. These results showed that beta1 integrin-mediated adhesion regulates Bim degradation and may contribute to the minimal residual disease associated with many haematopoietic malignancies. Together our data indicate that disrupting beta1 integrin-mediated regulation of Bim degradation may increase the efficacy of drugs, including imatinib, used to treat haematopoietic malignancies.

Nano-Fluidic Detection of Oncoprotein Signaling in Preclinical and Patient Lymphoma Samples.

Inactivation of oncogenes can be an effective cancer therapy. Determining precise levels of oncogene expression is important in the development of drugs to target oncogenes. We have developed a novel automated nano-fluidic Western-blot-like technology to detect and quantify oncogene expression in small numbers of mouse and human hematopoietic tumor cells. To detect different levels of oncogene expression, we generated transgenic mice in which the MYC or BCL2 oncogenes are regulated conditionally via the Tetracycline Regulatory System (Tet-system). Using lymphoma- derived cell lines from these mice, we titrated the level of oncogene expression by adding different concentrations of doxycycline in vitro. We were able to distinguish among different levels of oncogene expression in cell lysates, with high sensitivity in as few as 400 cells by nano-fluidic detection. Next, lymphoma derived cell lines were injected subcutaneously into syngeneic mice. Upon tumor development, MYC or BCL2 oncogenes were inactivated in vivo. Both MYC and BCL2 levels decreased in serial fine needle aspirations (FNAs) of tumor nodules upon parallel analysis with nano-fluidic detection and Western blot. Finally, we used nano-fluidic detection to determine levels of MYC, BCL2, AKT and ERK in lymph node samples from patients with follicular, transformed DLBC, Burkitt's, and mantle cell lymphomas. BCL2 was overexpressed in mantle cell and follicular lymphoma patients, confirmed by Western analysis, whereas MYC was found to be overexpressed in Burkitt's lymphoma. These results demonstrate that nano-fluidic detection technology may be used both as a preclinical tool for the assessment of changes in oncoprotein signaling and as a clinical diagnostic modality on microscopic clinical specimens.

C‐(2‐Chloroquinoline‐3‐yl)‐N‐phenyl Nitrone: New Synthetic Antioxidant Inhibits Proliferation and Induces Apoptosis of Breast Carcinoma MCF‐7 Cells

In this work, a new quinoline nitrone derivative, C-(2-chloroquinoline-3-yl)-N-phenyl nitrone (CQPN) was successfully prepared and proved by spectral analysis. The antioxidant activity of CQPN against various radicals was investigated and its anti-cancer properties against different human tumor cell lines including the solid tumor cell lines hepatocarcinoma (Hep-G2) and breast carcinoma (MCF-7); the hematopoietic tumor cell line lymphoblastic leukemia (1301) was also explored. CQPN activities were compared to that of the known nitrone C-phenyl-N-tert-butyl nitrone (PBN). Our results showed that although PBN was the stronger antioxidant than CQPN, the latter was an effective scavenger of different non-physiological (1,1-diphenyl-2-picrylhyrazyl) and physiological (peroxyl and hydroxyl) radicals. Both of CQPN and PBN possess a significant inhibitory property against LPS-stimulated NO production in macrophage. CQPN and PBN treatment resulted in a growth inhibition in Hep-G2 cells (IC50 31.42 microM and 18.6 microM, respectively). Unlike PBN, CQPN strongly inhibited the growth of MCF-7 cells (IC50 14.01 microM) in a dose-dependent manner. On contrary, CQPN and PBN exhibited a proliferative stimulatory activity of the immune cells including macrophages and lymphocytes. Exploring the cytotoxic effect of CQPN against MCF-7 cells indicated that CQPN led to a major time-dependent disturbance in the cell-cycle phases including progressive arrest in both S- and G2/M-phases. This disturbance was found to be associated with a kinetic induction of apoptosis. The novel nitrone derivative CQPN is a strong antioxidant, though less than PBN, and it may be an effective anti-proliferative compound against breast carcinoma.

CXCR4: a key receptor in the crosstalk between tumor cells and their microenvironment

Signals from the microenvironment have a profound influence on the maintenance and/or progression of hematopoietic and epithelial cancers. Mesenchymal or marrow-derived stromal cells, which constitute a large proportion of the non-neoplastic cells within the tumor microenvironment, constitutively secrete the chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). CXCL12 secretion by stromal cells attracts cancer cells, acting through its cognate receptor, CXCR4, which is expressed by both hematopoietic and nonhematopoietic tumor cells. CXCR4 promotes tumor progression by direct and indirect mechanisms. First, CXCR4 is essential for metastatic spread to organs where CXCL12 is expressed, and thereby allows tumor cells to access cellular niches, such as the marrow, that favor tumor-cell survival and growth. Second, stromal-derived CXCL12 itself can stimulate survival and growth of neoplastic cells in a paracrine fashion. Third, CXCL12 can promote tumor angiogenesis by attracting endothelial cells to the tumor microenvironment. CXCR4 expression is a prognostic marker in various types of cancer, such as acute myelogenous leukemia or breast carcinoma. Promising results in preclinical tumor models indicate that CXCR4 antagonists may have antitumor activity in patients with various malignancies. Collectively, these observations reveal that CXCR4 is an important molecule involved in the spread and progression of a variety of different tumors. As such, CXCR4 antagonists, although initially developed for treatment of AIDS, actually may become effective agents for the treatment of neoplastic disease.

Lenalidomide inhibits proliferation of Namalwa CSN.70 cells and interferes with Gab1 phosphorylation and adaptor protein complex assembly

Lenalidomide (Revlimid ®, CC-5013) belongs to a line of compounds known as immunomodulatory drugs (IMiDs ®) that are under clinical investigation in hematopoietic and solid tumor cancers. Lenalidomide efficacy has been reported in clinical trials of multiple myeloma and myelodysplastic syndromes (MDS), particularly in MDS patients with a del 5q cytogenetic abnormality, with or without other cytogenetic abnormalities. Here we report that lenalidomide inhibits proliferation of chromosome 5 deleted hematopoietic tumor cell lines in vitro, whether from the B cell, T cell, or myeloid lineage. There was diversity in the responses of the various cell lines to lenalidomide, with one undergoing cell cycle arrest, and others undergoing apoptosis. In the most lenalidomide-sensitive chromosome 5 deleted cell line, Namalwa CSN.70, the compound induced G 0/G 1 cell cycle arrest, inhibited Akt and Gab1 phosphorylation, and inhibited the ability of Gab1 to associate with a receptor tyrosine kinase. Lenalidomide also enhanced AP-1 transcriptional activity in Namalwa, but not in the other cell lines tested. These studies provide evidence for the mechanism of action of lenalidomide in chromosome 5 deleted hematopoietic tumors in vitro, and may provide a better understanding of the drug's activity in clinical applications.

Human GITR Ligand Is Expressed on Tumor Cells and Reduces Cytokine Production and Cellular Cytotoxicity of NK Cells Identified to Express GITR.

Members of the tumor necrosis factor (TNF) superfamily mediate multiple cellular functions including cellular proliferation, differentiation, and cell death. Human Glucocorticoid-induced TNF Receptor (GITR) has been shown to be expressed on T cells, is upregulated following activation and mediates costimulatory signals. The human GITR ligand (GITRL) has been reported to be expressed on antigen presenting cells and various healthy nonlymphoid tissues including small intestine, ovary, testis, kidney and endothelial cells. We analyzed multiple tumor cell lines of hematopoietic and epithelial origin as well as of germ cell lineage and various gliomas by RT-PCR and FACS analysis. Both GITRL m-RNA and protein are expressed in various carcinomas, gliomas and tumor cells of germ cell lineage, but not in hematopoietic tumor cells. Furthermore, we demonstrate that human NK cells constitutively express low levels of GITR, and this expression is upregulated following activation by, e.g., IL-2 or IL-15 as detected by quantitative PCR and FACS analysis. To address the functional interaction of GITRL with its receptor on NK cells, we generated a GITRL-IgG fusion protein (GITRL-Ig). Stimulation of activated NK cells with GITRL-Ig lead to significantly reduced IFN-g production of NK cells as measured by ELISA. Similarly, a significant reduction of IFN-g release was observed following coculture of GITR expressing NK cells with C1R cells transfected with GITRL but not with the respective mock transfectants. Furthermore, ligation of GITR on NK cells lead to significantly decreased killing of target cells as demonstrated by cellular cytotoxicity assays. Taken together, our data demonstrate that GITR not only plays an important role in adaptive immunity but is also involved in the regulation of NK cell effector functions. Since tumor cells express significant levels of GITRL, and ligation of GITR on NK cells markedly reduces cytokine production and cellular cytotoxicity, our data indicate that GITR-GITRL interactions play an important role in the escape of tumor cells from innate immune surveillance.

MicroRNA modulation of megakaryoblast fate involves cholinergic signaling

MicroRNAs (miRNAs) are abundant small regulatory RNAs with multiple roles in cell fate determination. The processes regulating cellular miRNA levels are still unclear and experimental oligonucleotide tools to readily mimic their effects are not yet available. Here, we report that thapsigargin-induced intracellular Ca ++ release suppressed pre-miR-181a levels in human promegakaryotic Meg-01 cells, induced differentiation-associated nuclear endoreduplication and caspase-3 activation and replaced the acetylcholinesterase 3′ splice variant AChE-S with AChE-R. AChE, PKC and PKA inhibitors all attenuated the pre-miR-181a decline and the induced differentiation. AChmiON, a synthetic 23-mer 2′-oxymethylated oligonucleotide mimicking the miR-181a sequence, blocked the calcium-induced differentiation while elevating cellular pre-miR-181a levels and inducing DNA fragmentation and cell death. Moreover, when added to RW 264.7 macrophages, AChmiON at 100 nM induced nitric oxide production with efficiency close to that of bacterial endotoxin, demonstrating physiologically relevant activities also in blood-born monocytes/macrophages. The stress-induced modulation of hematopoietic miR-181a levels through AChE, PKC and PKA cascade(s) suggests using miRNA mimics for diverting the fate of hematopoietic tumor cells towards differentiation and/or apoptosis.

Characterization of a novel, cytokine-inducible carboxypeptidase D isoform in haematopoietic tumour cells

CPD-N is a cytokine-inducible CPD (carboxypeptidase-D) isoform identified in rat Nb2 T-lymphoma cells. The prototypic CPD (180 kDa) has three CP domains, whereas CPD-N (160 kDa) has an incomplete N-terminal domain I but intact domains II and III. CPD processes polypeptides in the TGN (trans-Golgi network) but the Nb2 CPD-N is nuclear. The present study identified a cryptic exon 1', downstream of exon 1 of the rat CPD gene, as an alternative transcription start site that encodes the N-terminus of CPD-N. Western-blot analysis showed exclusive synthesis of the 160 kDa CPD-N in rat Nb2 and Nb2-Sp lymphoma cells. Several haematopoietic cell lines including human K562 myeloma, Jurkat T-lymphoma and murine CTLL-2 cytotoxic T-cells express a 160 kDa CPD-immunoreactive protein, whereas mEL4 T-lymphoma cells express the 180 kDa CPD. The CPD-immunoreactive protein in hK562 cells is also nuclear and cytokine-inducible. In contrast, MCF-7 breast cancer cells express only the 180 kDa CPD, which is mainly in the TGN. CPD/CPD-N assays using substrate dansyl-L-alanyl-L-arginine show approx. 98% of CPD-N activity in the Nb2 nucleus, whereas MCF-7 CPD activity is enriched in the post-nuclear 10000 g pellet. The K(m) for CPD-N and CPD are 132+/-30 and 63+/-9 microM respectively. Specific activity/K(m) ratios show that dansyl-L-alanyl-L-arginine is a better substrate for CPD-N than for CPD. CPD-N has an optimal pH of 5.6 (due to domain II), whereas CPD has activity peaks at pH 5.6 (domain II) and pH 6.5-7.0 (domain I). CPD and CPD-N are inhibited non-competitively by zinc chelator 1,10-phenanthroline and competitively by peptidomimetic inhibitor DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The Nb2 CPD-N co-immunoprecipitated with phosphatase PP2A (protein phosphatase 2A) and alpha4 phosphoprotein. In summary, a cytokine-inducible CPD-N is selectively expressed in several haematopoietic tumour cells. Nuclear CPD-N is enzymatically active and interacts with known partners of CPD.

Making Bad Cells Go Good: The Promise of Epigenetic Therapy

Among the prevailing explanations for the development of cancer over the past 50 years has risen the notion that clinically apparent cancers represent the failure of the tumor cells to differentiate along a pathway that would be expected for their tissues of origin. This point of view springs from and leads to the familiar characterization of epithelial neoplasms as well-, moderately, and poorly differentiated. This obviously orients our thinking about hematologic cancers, where the neoplastic cells frequently seem to be parodies of normal maturing marrow elements. Indeed, laboratory studies have demonstrated that exposure to various substances causes differentiation of certain hematopoietic tumor cells with their acquisition of more normal characteristics. Historically, the field received a tremendous boost—and a challenge—when Leder and Leder 1 demonstrated that high concentrations of sodium butyrate could cause marked differentiation of murine erythroleukemia cells. The implications that profound regulation of gene expression could be manipulated by so simple a molecule had tantalizing mechanistic and therapeutic implications. The subsequent demonstration that high concentrations of sodium butyrate caused marked change in the acetylation state of histones pointed toward modification of DNA or chromatin-associated proteins as correlating with the basis for this effect. 2 In parallel with these developments, longstanding observations that the methylation status of certain genes could influence their expression had arisen from the observations that there are actually five bases that could be detected in DNA. In addition to the familiar adenine, guanine, thymine, and cytosine, a variable proportion of cytosine existed as a methylated form. Moreover, the pattern of methylated cytosines was conserved from cycle to cycle of replication by the action of methyltransferases so that only one of the two chromosomes bearing certain genes was methylated. The methylated allele tended to be preferentially underexpressed. This nongenetic (in the sense that it did not arise from the sequence of the gene) but heritable feature was described as the mechanism ‘‘imprinting’’ certain maternally, as opposed to paternally, derived gene sets. 3 Methylation

411. Trapping of Adenovirus Vectors in Blood

In an attempt to circumvent the limitations of Adenovirus (Ad) vectors derived from group C serotype Ad5 we recently developed vectors based on group B serotype Ad11. These vectors are benefited by the low prevalence of Ad11-neutralizing antibodies in humans, and the CD46-dependent tropism of Ad11. CD46 is expressed at high levels in cell types including hematopoietic stem cells, dendritic cells and metastatic tumor cells. Following systemic intravenous delivery of an Ad5 vector, a chimeric Ad5 vector possessing the CD46 interacting Ad11 fiber (Ad5/11), and an Ad11 vector to CD46 transgenic mice we discovered that the kinetics of vector clearance from blood are different. Using real time quantitative PCR to detect viral genomes we found that the number of Ad11 vector genomes in mouse serum at 3 minutes post tail vein injection was 2 orders of magnitude lower than for Ad5 and Ad5/11 vectors, but at 15 and 120 minutes post injection the number of vector genomes was comparable between Ad5, Ad5/11 and Ad11. To investigate the differences between the numbers of vector genomes in serum at 3 minutes post injection the association of vector genomes with blood cells was also monitored by real time quantitative PCR at all time points. The number of vector genomes associated with blood cells was more than one order of magnitude higher for Ad11 than Ad5 or Ad5/11 at all time points tested with the highest difference seen at 3 minutes post injection (|[sim]|2 orders of magnitude higher). To study which type of blood cells associate with Ad11 vectors, CD46 transgenic mice were injected in the tail vein with 3H-[thymidine] labeled Ad vector. Individual cell types were separated from blood collected at 3 minutes post injection and the highest levels of tritium labeled Ad11 were associated with the platelet fraction. Subsequent analyses revealed that Ad5, Ad5/11 and Ad11 vectors associate with platelets within minutes after intravenous injection but that Ad11 vectors associate at higher levels. Electron microscopic analysis of platelets isolated 3 minutes after injection of Ad5, Ad5/11 or Ad11 vectors revealed that Ad particles were associated with the platelet surface and structures of the surface-connected canalicular system. We also noticed that Ad11 vectors associate with mononuclear cells more efficiently than Ad5/11 vectors. When taken together, the observations that Ad11 associates with platelets and mononuclear cells more efficiently than Ad5/11 suggests that capsid proteins other than fiber may mediate blood cell interactions. We are currently investigating the mechanism(s) by which Ad capsids and blood cells interact. The roles of the serotype specific (Ad5 or Ad11) capsid proteins hexon, penton and fiber in Ad/blood cell interactions will be presented. Our aim is to develop new Ad vectors that are not trapped in blood upon systemic intravascular administration.