Human Neoplastic Cells Research Articles

Death of human tumor endothelial cells in vitro through a probable calcium-associated mechanism induced by bevacizumab and detected via a novel method

AbstractWe isolated three dimensional cell clusters from fresh human solid tumors and also isolated human neoplastic and normal lymphatic cells. Cells were cultured for 96 hours with and without bevacizumab and other agents. At concentrations of bevacizumab which completely removed VEGF from the culture medium, dead microvascular cells were detected through Fast Green/H&E staining as previously described. These peculiar staining characteristics suggested the involvement of calcium, and this was confirmed through staining with Alizarin red S. Using Alizarin staining as a marker for endothelial cell death permitted the use of public domain image analysis software which resulted in a sensitive and specific system for identifying active pharmaceuticals which target the tumor microvasculature at the same time direct antitumor cell effects are determined. Our results suggest an important role for calcium in endothelial cell death mediated by bevacizumab and other agents and further suggest that agents promoting calcium influx may potentiate the activity of antiangiogenic agents.

The expression and activity of cystathionine-γ-lyase and 3-mercaptopyruvate sulfurtransferase in human neoplastic cell lines

The expression and activity of cystathionine γ-lyase (CST) and 3-mercaptopyruvate sulfurtransferase (MPST) were investigated in the human neoplastic cells lines: astrocytoma U373, neuroblastoma SH-SY5Y, melanoma A375, and melanoma WM35. Gene expression analysis demonstrated that the investigated neoplastic cells showed the expression of MPST and what is particularly interesting, the expression of CST. The presence of CST in these cells was confirmed using RT-PCR and western blot analysis. However, in U373 cells, a very low activity of CST was detected. In all the investigated cell lines, the activity of MPST was higher than that of CST, which suggests that in these cells, the main pathway of sulfane sulfur formation is the MPST-catalyzed reaction. RP-HPLC analysis showed a large disparity between the level of cystathionine and GSH in the investigated neoplastic cells. In SH-SY5Y cells, the low level of GSH and low GSH/GSSG ratio corresponded with the highest CST activity. Further investigations could aim at verifying whether the stimulation of CST, at the level of protein or gene expression, could change the proliferation of neoplastic cells.

PepT2 transporter protein expression in human neoplastic glial cells and mediation of fluorescently tagged dipeptide derivative β-Ala-Lys-Nε-7-amino-4-methyl-coumarin-3-acetic acid accumulation

The present study was aimed at analyzing the accumulation of the fluorescently tagged dipeptide derivative, beta-Ala-Lys-N(epsilon)-7-amino-4-methyl coumarin-3-acetic acid (AMCA), in primary cultures of human neoplastic glial cells. This molecule is a highly specific reporter used to investigate the dipeptide transport system hPepT2. In this study the authors used immunocytochemical methods to determine the cell-specific accumulation of a small and fluorescently tagged reporter molecule named beta-Ala-Lys-N(epsilon)-AMCA to detect dipeptide transport capacity of neoplastic glial cells. Furthermore, specific mRNA levels were quantified using Northern blot analysis and the tissue distribution of hPepT2 mRNA transcripts was demonstrated with in-situ hybridization histochemical analysis. Recent fluorescent immunocytochemical analyses have revealed that beta-Ala-Lys-N(epsilon)-AMCA specifically accumulates within anaplastic cells of astrocytic lineage but not in anaplastic oligodendrocytes or neurons. Northern blot analysis demonstrated that human hPepT2 mRNA is specifically detected in primary cell cultures of human glioblastoma but not in oligodendroglioma. Moreover, in situ hybridization analyses revealed an astrocytic localization of hPepT2 transcripts in human glioblastoma and astrocytoma cells. The hPepT2 transcription levels were clearly dependent on the grade of glial cell differentiation: within low-grade gliomas (WHO Grade II), more hPepT2 mRNA was detected compared with tumors of a higher grade of dedifferentiation (WHO Grade IV). Analysis of expression levels of hPepT2 mRNA in human neoplastic glial cells xenografted into the brains of athymic rats (han rnu(+/+)) showed a markedly increased expression of hPepT2 after 2 weeks of growth in vivo compared with the primary counterparts grown in vitro. The authors concluded that expression of the hPepT2 transporter protein is a characteristic of glial cells of astrocytic lineage, and is dependent on the grade of astroglial cell differentiation and the extracellular matrix (here brain neuropil). The authors found that beta-Ala-Lys-N(epsilon)-AMCA is as an excellent reporter molecule for assessing neoplastic glial cell function and physiological characteristics.

Calphostin C, a remarkable multimodal photodynamic killer of neoplastic cells by selective nuclear lamin B1 destruction and apoptogenesis (Review)

Perylenequinones that generate reactive oxygen species (ROS) when illuminated with visible light have been recommended as photodynamic chemotherapeutic agents. One of these is calphostin C (CalC), the action of the photo-activated derivative of which, CalCphiE, has been ascribed to its ability to selectively and irreversibly inhibit protein kinase Cs (PKCs). But recent results of experiments with neoplastic rat fibroblasts and human breast and uterine cervix cancer cells have revealed that the action of CalCphiE involves more than PKC inhibition. Besides suppressing PKC activity, CalCphiE rapidly causes endoplasmic reticulum (ER) stress in breast cancer cells and the selective complete oxidation and proteasomal destruction of the functionally essential nuclear envelope protein lamin B1, in human cervical carcinoma (HCC) cells and neoplastic rat fibroblasts. When these lamin B1-lacking cells are placed in the dark, cytoplasmic membrane-linked PKC activities suddenly rebound and apoptogenesis is initiated as indicated by the immediate release of cytochrome c from mitochondria and later on the activation of caspases. Hence, CalCphiE is a photodynamic cytocidal agent attacking multiple targets in cancer cells and it would be worth determining, even for their best applicative use, whether other perylenequinones also share the so far unexpectedly complex deadly properties of the CalCphiE.

Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells

AbstractThe mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL–negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34+ MPN cells than normal CD34+ HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.

Bosutinib Blocks Lyn and Btk Activation and Synergizes with the KIT D816V-Targeting Drug Midostaurin in Inducing Apoptosis in Neoplastic Human Mast Cells.

Abstract 1717Poster Board I-743Advanced systemic mastocytosis (SM) is a malignant hematopoietic neoplasm characterized by destructive growth of neoplastic mast cells (MC) in various organ systems. In these patients, the response to conventional cytoreductive therapy is poor and the prognosis is grave. The D816V-mutated variant of c-KIT is found in most patients and is considered to be a major transforming oncoprotein in SM that leads to abnormal survival and growth of neoplastic MC. Therefore, agents interfering with the kinase activity of KIT D816V have been developed. One promising agent is midostaurin (PKC412). However, in most patients with advanced SM, therapy with midostaurin is not sufficient to induce long term remissions. In addition, midostaurin is unable to block all pro-oncogenic signaling molecules, such as Lyn and Btk, in neoplastic MC, suggesting that additional oncoproteins and survival factors may play a role in malignant transformation in SM, and that novel therapeutic strategies are required to block such KIT-independent oncogenic pathways. Especially Lyn and Btk have attracted attention as potential new targets in neoplastic MC. Bosutinib (SKI-606) is a novel multikinase inhibitor that targets a broad spectrum of kinases including Lyn and Btk. The aim of the current study was to evaluate the effect of bosutinib on neoplastic MC, and potential cooperative drug interactions between bosutinib and midostaurin. As assessed by 3H-thymidine uptake, bosutinib was found to inhibit the growth of the MC leukemia cell line HMC-1, including the HMC-1.1 subclone that lacks KIT D816V and HMC-1.2 cells expressing KIT D816V, with similar IC50 values (1-5 μM). Furthermore, bosutinib was found to induce apoptosis in both HMC-1 subclones. Growth-inhibitory and apoptosis-inducing effects of bosutinib were also seen in primary neoplastic MC obtained from the bone marrow of patients with SM (n=3). As assessed by phosphoblotting, bosutinib did not inhibit the autophosphorylation of mutant KIT in HMC-1 cells, but was found to completely inhibit the phosphorylation of Lyn and Btk. To confirm the target-function of Lyn and Btk in neoplastic MC, siRNA experiments were performed. Knockdown of Lyn or Btk resulted in induction of apoptosis and growth-inhibition in HMC-1 cells. We next attempted to exploit target-specific and complementing effects of midostaurin and bosutinib by combining both substances. As expected, combined application of bosutinib and midostaurin resulted in a complete inhibition of phosphorylation of KIT, Lyn, and Btk in HMC-1.1 and HMC-1.2 cells. We were also able to show that bosutinib synergizes with midostaurin in inducing apoptosis in both HMC-1 subclones. Synergistic effects were also observed when combining midostaurin with Lyn- or Btk-siRNA. Together, we have identified Lyn and Btk as novel KIT-independent survival molecules in neoplastic MC. Inhibition of these kinases by siRNA-knockdown or by bosutinib leads to growth-inhibition and apoptosis. Synergistic pro-apoptotic effects were observed with the combination “bosutinib + midostaurin”, suggesting that simultaneous targeting of KIT and Lyn/Btk may be a powerful strategy to counteract the survival of neoplastic MC. This drug combination may therefore be an interesting approach to overcome drug-resistance in advanced forms of SM. DisclosuresValent:Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Identification of Oncostatin M as a Novel KIT D816V-Dependent Cytokine in Neoplastic Human Mast Cells.

Abstract 213Systemic mastocytosis (SM) is a neoplastic disease of mast cells (MC) and their bone marrow-derived progenitors. The clinical picture in SM is variable ranging from an indolent course to highly aggressive variants with short survival time. The pathologic hallmark in SM is the multifocal dense infiltrate of MC in the bone marrow. Other typical features of SM include alterations of the bone marrow microenvironment such as increased angiogenesis and fibrosis. In a majority of patients, MC display the KIT mutation D816V which affects the activation loop at the entrance to the enzymatic pocket of the KIT kinase. As a consequence, KIT D816V exhibits constitutive tyrosine kinase activity and promotes cytokine-independent differentiation of MC. However, so far, little is known about KIT D816V-dependent expression of pathogenetically relevant molecules in neoplastic MC. Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin-6 family which is produced mainly by activated T cells and monocytes. OSM has been shown to inhibit cell growth in cell lines derived from solid tumors but to stimulate proliferation of fibroblasts and endothelial cells. Recently, it has been reported that OSM produced by activated MC promotes growth of human dermal fibroblasts. Moreover, it has been suggested that OSM stimulates growth of murine bone marrow-derived mast cells in a mast cell/fibroblast coculture. However, expression of OSM in neoplastic MC or a potential pathogenetic role of OSM in SM have not been examined so far. The aim of the present study was to analyze expression of OSM in neoplastic human MC and to determine the role of KIT D816V in OSM expression. As assessed by immunohistochemistry performed on bone marrow sections of patients with SM, typical spindle-shaped neoplastic MC were found to express OSM. Serial section-staining confirmed that tryptase-positive MC co-express OSM. Expression of OSM was found in neoplastic MC in all patients investigated (n=15) and in all variants of SM (indolent SM as well as aggressive variants) with comparable staining intensities. Preincubation of anti-OSM antibody with a specific blocking peptide resulted in a negative stain. In Ba/F3 cells, doxycycline-inducible expression of KIT D816V led to a substantial upregulation of OSM mRNA and OSM protein, whereas expression of wild type KIT did not affect expression of OSM. In addition, the KIT D816V-positive HMC-1.2 mast cell line was found to express OSM at high levels, whereas the KIT D816V-negative HMC-1.1 subclone expressed only baseline levels of OSM. Correspondingly, the KIT D816V-targeting drug midostaurine (PKC412) decreased the expression of OSM in HMC-1.2 cells as well as in KIT D816V-expressing Ba/F3 cells in a dose-dependent manner. To investigate signaling pathways involved in KIT D816V-dependent expression of OSM, we applied pharmacologic inhibitors and dominant negative-acting signaling molecules. We found that KIT D816V-dependent expression of OSM is inhibited by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Expression of dominant negative mutants of signal transducer and activator of transcription 5 (STAT5) did not affect expression of OSM in KIT D816V-expressing cells. In summary, our data identify OSM as a novel cytokine expressed in neoplastic MC in patients with SM and show that KIT D816V directly promotes expression of OSM through activation of the mitogen-activated protein-kinase pathway. OSM may be an important KIT D816V-dependent effector promoting angiogenesis and fibrogenesis/sclerosis in patients with SM. Disclosures:No relevant conflicts of interest to declare.

Immunohistochemical expression of aromatase and estrogen, androgen and progesterone receptors in normal and neoplastic human meningeal cells

Evidence suggests that sex hormones may play a role in the tumorigenesis of meningiomas, and studies have demonstrated the expression of hormone receptors in these tumors. Aromatase expression has been detected in several normal tissues, including neurons in the CNS, and tumor tissues. We aim to assess the expression of aromatase (ARO) and of progesterone receptor (PR), estrogen receptor (ER) and androgen receptor (AR) in both normal and neoplastic meningeal cells. A cross-sectional study was conducted with 126 patients diagnosed with meningioma (97 women and 29 men; mean age, 53.6 years) submitted to neurosurgery at Hospital São José, Complexo Hospitalar Santa Casa de Porto Alegre, southern Brazil. Control sections of normal meningeal cells, 19 patients, were obtained by evaluating the arachnoid tissue present in the arachnoid cyst resected material. Immunohistochemistry was applied to assess ARO, PR, ER and AR. Aromatase expression was detected in 100% of the control patients and in 0% of the patients with meningioma. ER was present in 24.6% of the meningiomas and in 0% of the controls, AR in 18.3% of the meningiomas and in 0% of the controls, and PR in 60.3% of the meningiomas and in 47.4% of the controls. A positive association was observed between the presence of AR and ER (OR 3.7; P = 0.01) in meningiomas. There were no significant differences in the presence of hormone receptors between meningioma histological subtypes. PR expression in women with meningioma was significantly higher than that found in men (OR 2.3; P = 0.08). Behavior pattern differences observed between aromatase expression, present in normal tissues and absent in meningiomas, and estrogen and androgen hormone receptors, absent in normal tissues and present in meningiomas, suggest that there is heterogeneity in modulation by sex steroids in the development of these tumors.

Studies on the production of actinomycin-D byStreptomyces griseoruber- a novel source

To isolate and characterize bioactive metabolites produced by a micro-organism isolated from a soil sample associated with the roots of a medicinal plant, Azadirachta indica. Morphological, cultural, physiological and 16S rRNA homology studies revealed that the organism showed 99% similarity with Streptomyces griseoruber NBRC 12873. One bioactive metabolite (Py2) isolated from the fermented broth was characterized as actinomycin-D (act-D). It showed high activity against various gram-positive and gram-negative bacterial cultures, Mycobacterium tuberculosis H37Rv and human neoplastic cells in vitro using standard protocols. The isolated strain S. griseoruber produced act-D predominantly (210 mg l(-1), c. 88% of the crude) under nonoptimized growth conditions. Streptomyces griseoruber may be exploited as a potential source for the commercial production of act-D, as this strain is not reported to produce act-D. Further investigations on the strain for commercial application will be of immense pharmaceutical importance.

Identification of a normal human bone marrow cell population co-expressing megakaryocytic and erythroid markers in culture.

A population of haematopoietic cells co-expressing glycoprotein IIIa (GPIIIa), which has been shown to be present in the megakaryocyte-platelet lineage, and glycophorin A, which has been shown to be specific for the erythroid lineage, has been identified in normal bone marrow cultures using double immunofluorescence staining. The cells showing this phenotype have the size of lymphocytes and appear at an early time (day 1 to d 6) of culture. We have also detected a type of mixed megakaryocyte (MK) cluster containing this phenotype on d 3 or 4 in most normal marrow samples. Such a cell phenotype was not detected after 6 d of culture. It is thus the first time that this phenotype, although already described for several human neoplastic cell lines, has been observed in several normal human bone marrow cultures. Cells expressing this phenotype may represent haematopoietic cells, common to megakaryocytic and erythroid lineages, at a very early stage of differentiation.

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 +/- 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4-23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.

Alkyl-capped silicon nanocrystals lack cytotoxicity and have enhanced intracellular accumulation in malignant cells via cholesterol-dependent endocytosis.

Nanocrystals of various inorganic materials are being considered for application in the life sciences as fluorescent labels and for such therapeutic applications as drug delivery or targeted cell destruction. The potential applications of the nanoparticles are critically compromised due to the well-documented toxicity and lack of understanding about the mechanisms involved in the intracellular internalization. Here intracellular internalization and toxicity of alkyl-capped silicon nanocrystals in human neoplastic and normal primary cells is reported. The capped nanocrystals lack cytotoxicity, and there is a marked difference in the rate and extent of intracellular accumulation of the nanoparticles between human cancerous and non-cancerous primary cells, the rate and extent being higher in the malignant cells compared to normal human primary cells. The exposure of the cells to the alkyl-capped nanocrystals demonstrates no evidence of in vitro cytotoxicity when assessed by cell morphology, apoptosis, and cell viability assays. The internalization of the nanocrystals by Hela and SW1353 cells is almost completely blocked by the pinocytosis inhibitors filipin, cytochalasin B, and actinomycin D. The internalization process is not associated with any surface change in the nanoparticles, as their luminescence spectrum is unaltered upon transport into the cytosol. The observed dramatic difference in the rate and extent of internalization of the nanocrystals between malignant and non-malignant cells therefore offers potential application in the management of human neoplastic conditions.

Comprehensive mutational analysis and mRNA isoform quantification of TP63 in normal and neoplastic human prostate cells

The role of TP63 in cancer remains controversial since both oncogenic and tumor suppressive actions have been reported. p63 protein is found in the nuclei of basal cells of the normal prostate, yet it is absent in the vast majority of prostate cancer nuclei. Since a complex array of TP63 mRNA transcripts encode polypeptides with distinct functional properties, it is important to determine which forms are expressed in normal and prostate cancer tissue. We used real-time RT-PCR to distinguish TP63 mRNA isoforms in prostate cancer cell lines (n = 7), and samples from prostate cancer patients. We sequenced all TP63 exons from 20 primary tumors, 20 metastases, 28 tumor xenografts, and 7 prostate cancer cell lines. TP63 mRNA isoforms were present in all tumors, albeit at levels lower than in normal prostate. The most abundant N-terminal variant was DeltaN; the most abundant C-terminal variant was the alpha form. The prostate tumor cell line CWR22Rv1 contained a single G to T substitution in exon 8 that is identical to a dominant-negative DNA binding inactivation mutation occurring in patients with a congenital TP63 deficiency syndrome. One patient tumor contained a somatic mutation in exon 11. The pattern of TP63 mRNA expression in normal prostate tissue is retained in reduced amounts in prostate cancer, and a potentially functional TP63 mutation was identified in one prostate tumor. Thus, if TP63 is a prostate cancer gene it likely functions as a tumor suppressor. Further study of the role of TP63 isoforms in regulating stem cell functions of normal and neoplastic prostate epithelial cells is needed. Prostate 69:559-569, 2009. (c) 2009 Wiley-Liss, Inc.

Identification and bioinformatic characterization of a multidrug resistance associated protein (ABCC) gene in Plasmodium berghei

BackgroundThe ATP-binding cassette (ABC) superfamily is one of the largest evolutionarily conserved families of proteins. ABC proteins play key roles in cellular detoxification of endobiotics and xenobiotics. Overexpression of certain ABC proteins, among them the multidrug resistance associated protein (MRP), contributes to drug resistance in organisms ranging from human neoplastic cells to parasitic protozoa. In the present study, the Plasmodium berghei mrp gene (pbmrp) was partially characterized and the predicted protein was classified using bioinformatics in order to explore its putative involvement in drug resistance.MethodsThe pbmrp gene from the P. berghei drug sensitive, N clone, was sequenced using a PCR strategy. Classification and domain organization of pbMRP were determined with bioinformatics. The Plasmodium spp. MRPs were aligned and analysed to study their conserved motifs and organization. Gene copy number and organization were determined via Southern blot analysis in both N clone and the chloroquine selected line, RC. Chromosomal Southern blots and RNase protection assays were employed to determine the chromosomal location and expression levels of pbmrp in blood stages.ResultsThe pbmrp gene is a single copy, intronless gene with a predicted open reading frame spanning 5820 nucleotides. Bioinformatic analyses show that this protein has distinctive features characteristic of the ABCC sub-family. Multiple sequence alignments reveal a high degree of conservation in the nucleotide binding and transmembrane domains within the MRPs from the Plasmodium spp. analysed. Expression of pbmrp was detected in asexual blood stages. Gene organization, copy number and mRNA expression was similar in both lines studied. A chromosomal translocation was observed in the chloroquine selected RC line, from chromosome 13/14 to chromosome 8, when compared to the drug sensitive N clone.ConclusionIn this study, the pbmrp gene was sequenced and classified as a member of the ABCC sub-family. Multiple sequence alignments reveal that this gene is homologous to the Plasmodium y. yoelii and Plasmodium knowlesi mrp, and the Plasmodium vivax and Plasmodium falciparum mrp2 genes. There were no differences in gene organization, copy number, or mRNA expression between N clone and the RC line, but a chromosomal translocation of pbmrp from chromosome 13/14 to chromosome 8 was detected in RC.

Effects of the Mcl-1/Bcl-2 Inhibitor GX015-070 (Obatoclax®) on Growth and Viability of Canine and Human Neoplastic Mast Cells

Mcl-1 is a Bcl-2 family-member that has been described to act anti-apoptotic in various myeloid neoplasms. We and others have recently shown that neoplastic mast cells (MC) in patients with systemic mastocytosis (SM) display Mcl-1, Bcl-2, and Bcl-xL. In the present study, we examined the effects of the Mcl-1/Bcl-2-targeting drug GX015-070 (obatoclax®; GeminX, Montréal, Quebéc, Canada) on growth and viability of primary neoplastic MC obtained from patients with SM (n=3), the human MC leukemia cell line HMC-1, and the canine mastocytoma cell line C2. Two HMC-1 subclones, one lacking KIT D816V (HMC- 1.1) and one expressing KIT D816V (HMC-1.2) were examined. As assessed by RT-PCR and immunostaining, primary neoplastic MC as well as HMC-1 cells (both subclones) were found to express Mcl-1 mRNA and the Mcl-1 protein in a constitutive manner, but did not express significant amounts of proapoptotic Bim. Transfection of HMC-1 cells with Mcl-1-specific siRNA resulted in reduced proliferation and increased apoptosis compared to cells transfected with a control siRNA. GX015-070 was found to inhibit 3H-thymidine uptake and thus proliferation in HMC-1 cells in a dose-dependent manner, with higher IC50 values obtained in HMC-1.2 cells (0.5 μM) compared to HMC-1.1 cells (0.05 μM). GX015-070 also inhibited the growth and survival in the canine mastocytoma cell line C2 (IC50: 0.5-1 μM). Moreover, GX015-070 was found to inhibit the proliferation of primary human neoplastic MC in all SM patients tested (IC50: 0.05-0.1 μM). We next attempted to combine obatoclax with a modulator of Mcl-1/Bim expression in MC, in order to enhance drug effects. Since Bim is degraded via the proteasome, we applied the proteasome inhibitor bortezomib. Whereas GX015-07 did not modulate the production/expression of Mcl-1 or Bim in HMC-1 cells, bortezomib was found to promote the expression of Bim in our Western blot experiments. In addition, bortezomib was found to suppress 3H-thymidine uptake in both HMC-1 subclones. Finally, bortezomib was found to cooperate with GX015-070 in producing apoptosis in HMC-1.1 cells, HMC-1.2 cells, and C2 cells. Together, our data show that the Mcl-1/Bcl-2-targeting drug GX015-070 is a potent inhibitor of in vitro growth and survival of canine and human neoplastic MC. Targeting of Mcl-1 in neoplastic MC alone or in combination with a Bim-regulator may be an interesting pharmacologic approach in advanced SM.

104 POSTER Role of P21 in sensitivity to DACH-platinum compounds, oxaliplatin and ProLindac, in human cancer cells

In order to clarify the relation between the reactivity of the leaving groups in cisplatin-like complexes and their activity/toxicity, six new complexes of the formula Pt(NH3)2X and Pt(dach)X (where X is selenato, anion of squaric acid, or demethylcantharic acid) have been synthesized and compared. These complexes have been characterized by elemental analysis, infrared spectroscopy, and conductivity measurements. Six human neoplastic cell lines (HCT, KB, BGC, HL-60, K-562 and Bel-7402) were used to screen these compounds. The results demonstrated that four of them have comparable IC50 and even lower IC50 in a few kinds of tumor cell lines compared to cisplatin. Their LD50 values showed that the toxicity of these platinum complexes is related to the reactivity of the leaving groups. All of these complexes have lower acute toxicity than cisplatin, but the anticancer activity is not affected.

Redox mechanisms switch on hypoxia-dependent epithelial–mesenchymal transition in cancer cells

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.

Lactate dehydrogenase (LDH) isoenzymes and proliferative activity of lymphoid cells—an immunocytochemical study

In a recent immunohistochemical study, we suggested that elevation of LDH isoenzymes is generally related to cell proliferation. To explore this relationship further, we have now examined the expression of H- and M-type LDH isoenzymes immunocytochemically in human resting and mitogen-activated B and T lymphocytes. In the resting state, T lymphocytes showed strong staining for H-type LDH but showed little or no staining for M-type LDH, while B lymphocytes showed only weak staining for M-type LDH. During activation of T cells, M-type LDH started to increase when cells entered the early stages of the cell cycle. The staining intensity increased to a maximum when the percentage of the T cells at the S/G2/M phases of the cell cycle reached its peak. M-type LDH expression declined when the activated T cells returned to their resting state. Staining for H-type LDH remained strong in T cells during activation. In B lymphocytes, both H- and M-type LDH isoenzymes increased concomitantly following activation and the staining intensity also correlated well with the percentage of the S/G2/M fraction. The expression of H- and M-type LDH was also determined in fresh leukaemia and a variety of lymphoid cell lines. It was noted that cells of chronic lymphocytic leukaemia (CLL) and pro-lymphocytic leukaemia (PLL), morphologically similar to normal lymphocytes, showed a LDH staining pattern resembling that of resting B lymphocytes, while lymphoblasts in T cell acute lymphoblastic leukaemia (T-ALL), high grade B cell lymphoma and Epstein-Barr virus (EBV) transformed cell lines showed a LDH staining pattern similar to that in activated T or B lymphocytes. Taken together, our results have demonstrated a significant correlation between expression of LDH and proliferative activity of cells. Immunostaining with the MoAbs to H- and M-type LDH can, therefore, provide a useful means not only for identification of T and B lymphocytes but also for rapid evaluation of the proliferating fraction of normal and neoplastic human cell populations.

Luminal regulation of normal and neoplastic human EC cell serotonin release is mediated by bile salts, amines, tastants, and olfactants

Mechanisms by which gut luminal content regulates secretion and motility are ill understood. We evaluated whether neuroendocrine enterochromaffin (EC) cells act as luminal sensors for a wide variety of nutrients and defined the secretory mechanisms of this process. Pure (98-99%) FACS-sorted human EC cells and neoplastic EC cells (KRJ-I) were studied. RT-PCR identified transcripts for T2R1 (bitter), OR1G1 (class II olfactory) and trace amine (TAR1) G protein-coupled receptors (GPCRs) and transporters for glutamine (SNAT1/2), glucose (GLUT1/3/SGLT1), and bile salts (ABST). Glutamine and sodium deoxycholate stimulated 5-HT release (EC(50) = 0.002-0.2 microM; 2-fold release) but were 10-100 times more potent in neoplastic EC cells, which also secreted 6-13 times more 5-HT. Tastants (caffeine, tyramine, octopamine) and olfactants (thymol and eugenol) also stimulated normal and neoplastic EC cell 5-HT secretion (EC(50) = 1.2 nM to 2.1 microM and 0.05 nM to 0.1 microM release, respectively); 2-deoxyglucose and the artificial sweetener sucralose also stimulated (EC(50) = 9.2 and 0.38 nM). 5-HT release was associated with ERK phosphorylation (1.5-fold, P < 0.02) and could be inhibited by a somatostatin analog (IC(50) = 1 pM). Eleven secretory associated genes including the vesicle docking inhibitor STXBP3 were upregulated in response to glutamine and bile salt stimulation in neoplastic EC cells. Targeting STXBP3 expression by use of antisense knockdown significantly (P < 0.05) reduced 5-HT secretion. In conclusion, EC cells express GPCRs and transporters for luminal tastants, olfactants, glutamine, glucose, and bile salts. Activation includes a panel of secretory genes, ERK phosphorylation, and 5-HT secretion. Luminal EC cell regulation is likely to be as important as G cell regulation in gastric acid secretion; development of agents to target EC cell function is therefore a critical therapeutic goal.

Generation and Characterization of Monoclonal Antibodies to NIAM: A Nuclear Interactor of ARF and Mdm2

Nuclear interactor of ARF and Mdm2 (NIAM) is a newly discovered growth inhibitor that helps maintain chromosomal stability. It is functionally linked to the ARF-Mdm2-p53 tumor suppressor pathway and is predicted to be a tumor suppressor, but the lack of antibodies capable of detecting the endogenous human protein has delayed efforts to define its role in human tumorigenesis. This study reports the development, screening, and characterization of several monoclonal antibodies (MAbs) that specifically recognize endogenous human NIAM protein by Western blotting, immunoprecipitation, immunofluorescence, and immunohistochemistry. These MAbs are predicted to be important tools for evaluating the expression and physiological function of NIAM in normal versus neoplastic human cells and tissues.