In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

In vitro proliferation and differentiation of hepatic oval cells and their potential capacity for intrahepatic transplantation

Hepatic oval cells (HOCs) are recognized as facultative liver progenitor cells that play a role in liver regeneration after acute liver injury. Here, we investigated the in vitro proliferation and differentiation characteristics of HOCs in order to explore their potential capacity for intrahepatic transplantation. Clusters or scattered HOCs were detected in the portal area and interlobular bile duct in the liver of rats subjected to the modified 2-acetylaminofluorene and partial hepatectomy method. Isolated HOCs were positive for c-kit and CD90 staining (99.8% and 88.8%, respectively), and negative for CD34 staining (3.6%) as shown by immunostaining and flow cytometric analysis. In addition, HOCs could be differentiated into hepatocytes and bile duct epithelial cells after leukemia inhibitory factor deprivation. A two-cuff technique was used for orthotopic liver transplantation, and HOCs were subsequently transplanted into recipients. Biochemical indicators of liver function were assessed 4 weeks after transplantation. HOC transplantation significantly prolonged the median survival time and improved the liver function of rats receiving HOCs compared to controls (P=0.003, Studentt-test). Administration of HOCs to rats also receiving liver transplantation significantly reduced acute allograft rejection compared to control liver transplant rats 3 weeks following transplantation (rejection activity index score: control=6.3±0.9; HOC=3.5±1.5; P=0.005). These results indicate that HOCs may be useful in therapeutic liver regeneration after orthotopic liver transplantation.

A combination of STI571 and BCR-ABL1 siRNA with overexpressed p15INK4B induced enhanced proliferation inhibition and apoptosis in chronic myeloid leukemia

p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.

The consensus sequence of FAMLF alternative splice variants is overexpressed in undifferentiated hematopoietic cells

The familial acute myeloid leukemia related factor gene (FAMLF) was previously identified from a familial AML subtractive cDNA library and shown to undergo alternative splicing. This study used real-time quantitative PCR to investigate the expression of the FAMLF alternative-splicing transcript consensus sequence (FAMLF-CS) in peripheral blood mononuclear cells (PBMCs) from 119 patients with de novo acute leukemia (AL) and 104 healthy controls, as well as in CD34+cells from 12 AL patients and 10 healthy donors. A 429-bp fragment from a novel splicing variant of FAMLF was obtained, and a 363-bp consensus sequence was targeted to quantify total FAMLF expression. Kruskal-Wallis, Nemenyi, Spearman's correlation, and Mann-Whitney U-tests were used to analyze the data. FAMLF-CS expression in PBMCs from AL patients and CD34+ cells from AL patients and controls was significantly higher than in control PBMCs (P<0.0001). Moreover,FAMLF-CS expression in PBMCs from the AML group was positively correlated with red blood cell count (rs=0.317, P=0.006), hemoglobin levels (rs=0.210, P=0.049), and percentage of peripheral blood blasts (rs=0.256, P=0.027), but inversely correlated with hemoglobin levels in the control group (rs=–0.391, P<0.0001). AML patients with high CD34+ expression showed significantly higherFAMLF-CS expression than those with low CD34+ expression (P=0.041). Our results showed thatFAMLF is highly expressed in both normal and malignant immature hematopoietic cells, but that expression is lower in normal mature PBMCs.

Innate immunity genes as determinants of resistance/susceptibility to human disease : studies in leukemia patients

La leucémie lymphoblastique aiguë des cellules Pré-B (B-ALL) reste le type de cancer le plus souvent diagnostiqué chez les enfants. Des études ont montré que des déterminants génétiques jouent un rôle important dans la susceptibilité/résistance au développement de ce cancer. À cet égard, les gènes Killer-cell Immunoglobulin-like Receptor (KIR) sont d'une importance particulière. Ces gènes sont fortement polymorphiques et codent pour des récepteurs qui contrôlent l’activité fonctionnelle des cellules Natural Killer (NK). Notre hypothèse est que les gènes activateurs des KIR s’associent avec la résistance innée pour développer la B-ALL. Afin d'évaluer cette hypothèse, nous avons entrepris une étude de cas-contrôles chez des enfants canadiens-français dans laquelle nous avons utilisé l'ADN génomique de 100 patients atteints de B-ALL ainsi que l’ADN de 245 individus sains. La présence ou l'absence de chaque gène KIR a été détectée par PCR en utilisant des amorces de séquences spécifiques. Nous avons trouvé que la présence des gènes KIR activateurs est significativement diminuée chez les enfants leucémiques par rapport aux témoins. En outre, le nombre de ces gènes a aussi montré une association significative linéaire avec la résistance au développement d’une B-ALL. Cela suggère des effets additifs de ces gènes permettant de conférer une protection contre ce cancer. Ces résultats pourraient être utiles afin de déceler de façon précoce les enfants ayant un risque de développer cette leucémie. Enfin, des stratégies thérapeutiques basées sur les récepteurs KIR pourraient être envisagées et s'avérer utiles concernant le traitement de ce cancer chez les enfants.

Large scale identification of protein SUMOylation by mass spectrometry in HEK293 cells

Les fichiers qui accompagnent mon document sont des tableaux supplémentaires réalisés avec Excel (Microsoft Office), dans la version papier du mémoire ces fichiers sont sur un CD-ROM.

Understanding the transcriptional control of EIF4E and its dysregulation in acute myeloid leukemia: role of NF-κB

EIF4E, le facteur d’initiation de la traduction chez les eucaryotes est un oncogène puissant et qui se trouve induit dans plusieurs types de cancers, parmi lesquels les sous-types M4 et M5 de la leucémie aiguë myéloblastique (LAM). EIF4E est régulé à plusieurs niveaux cependant, la régulation transcriptionnelle de ce gène est peu connue. Mes résultats montrent que EIF4E est une cible transcriptionnelle directe du facteur nucléaire « kappa-light- chain- enhancer of activated B cells » (NF-κB).Dans les cellules hématopoïétiques primaires et les lignées cellulaires, les niveaux de EIF4E sont induits par des inducteurs de NF-κB. En effet, l’inactivation pharmaceutique ou génétique de NF-κB réprime l’activation de EIF4E. En effet, suite à l’activation de NF-κB chez l’humain, le promoteur endogène de EIF4E recrute p65 (RelA) et c-Rel aux sites évolutionnaires conservés κB in vitro et in vivo en même temps que p300 ainsi que la forme phosphorylée de Pol II. De plus, p65 est sélectivement associé au promoteur de EIF4E dans les sous-types LAM M4/M5 mais non pas dans les autres sous-types LAM ou dans les cellules hématopoïétiques primaires normales. Ceci indique que ce processus représente un facteur essentiel qui détermine l’expression différentielle de EIF4E dans la LAM. Les analyses de données d’expressions par séquençage de l’ARN provenant du « Cancer Genome Atlas » (TCGA) suggèrent que les niveaux d’ARNm de EIF4E et RELA se trouvent augmentés dans les cas LAM à pronostic intermédiaire ou faible mais non pas dans les groupes cytogénétiquement favorables. De plus, des niveaux élevés d’ARNm de EIF4E et RELA sont significativement associés avec un taux de survie relativement bas chez les patients. En effet, les sites uniques κB se trouvant dans le promoteur de EIF4E recrutent le régulateur de transcription NF-κB p65 dans 47 nouvelles cibles prévues. Finalement, 6 nouveaux facteurs de transcription potentiellement impliqués dans la régulation du gène EIF4E ont été prédits par des analyses de données ChIP-Seq provenant de l’encyclopédie des éléments d’ADN (ENCODE). Collectivement, ces résultats fournissent de nouveaux aperçus sur le control transcriptionnel de EIF4E et offrent une nouvelle base moléculaire pour sa dérégulation dans au moins un sous-groupe de spécimens de LAM. L’étude et la compréhension de ce niveau de régulation dans le contexte de spécimens de patients s’avère important pour le développement de nouvelles stratégies thérapeutiques ciblant l’expression du gène EIF4E moyennant des inhibiteurs de NF-κB en combinaison avec la ribavirine.

TAF15 and the leukemia-associated fusion protein TAF15-CIZ/NMP4 are cleaved by caspases-3 and-7

Caspases are central players in proteolytic pathways that regulate cellular processes Such as apoptosis and differentiation. To accelerate the discovery of novel caspase substrates we developed a method combining in silico screening and in vitro validation. With this approach, we identified TAH15 as a novel caspase Substrate in a trial Study. We find that TAF15 was specifically cleaved by caspases-3 and -7. Site-directed mutagenesis revealed the consensus sequence (106)DQPD/Y(110) as the only site recognized by these caspases. Surprisingly, TAF15 was cleaved at more than one site in staurosporine-treated Jurkat cells. In addition, we generated two oncogenic TAF15-CIZ/NMP4-fused proteins which have been found in acute myeloid leukemia and demonstrate that caspases-3 and -7 cleave the fusion proteins at one single site. Broad application of this combination approach should expedite identification of novel caspase-interacting proteins and provide new insights into the regulation of caspase pathways leading to cell death in normal and cancer cells. (C) 2009 Elsevier Inc. All rights reserved.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

Interleukin-4 and interleukin-13 inhibit the expression of leukemia inhibitory factor and interleukin-11 in fibroblasts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

B lineage acute lymphoblastic leukemia transformation in a child with juvenile myelomonocytic leukemia, type 1 neurofibromatosis and monosomy of chromosome 7 - Possible implications in the leukemogenesis

This report describes the case of an 8-month-old infant with a diagnosis of juvenile myelomonocytic leukemia (JMML) and type I neurofibromatosis that presented progression to B lineage acute lymphoid leukemia (ALL). The same rearrangement of gene T-cell receptor gamma (TCRgamma) was detected upon diagnosis of JMML and ALL, suggesting that both neoplasias may have evolved from the same clone. Our results support the theory that JMML may derive from pluripotential cells and that the occurrence of monosomy of chromosome 7 within a clone of cells having an aberrant neurofibromatosis type 1 (NFI) gene may be the cause of JMML and acute leukemia. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Apoptosis in the epithelial cells of the rests of Malassez of the periodontium of rat molars

Background and Objectives: Epithelial rests of Malassez are clusters of cells derived from Hertwig's root sheath that remain in the periodontal ligament throughout life. Although it is known that the cells of Malassez proliferate, there are no studies showing that they undergo programmed cell death, i.e. apoptosis. In most tissues, proliferation is balanced by apoptosis. Thus we examined regions of the periodontium of young and adult rat molars in the hope of detecting apoptosis.Methods: Wistar rats aged 29, 45 and 120 days were killed with chloral hydrate (600 mg/kg). Fragments containing maxillary molars were removed and fixed in formaldehyde, decalcified, and embedded in paraffin and glycol methacrylate. Sections were stained with hematoxylin/eosin and the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) method for detection of apoptosis. Specimens were also fixed in glutaraldehyde-formaldehyde, decalcified and processed for transmission electron microscopy.Results: Epithelial rests of Malassez containing round/ovoid basophilic dense bodies and TUNEL-positive structures were found in all specimens examined. Ultrastructural examination revealed that some cells of Malassez contained masses of condensed peripheral chromatin and a shrunken cytoplasm exhibiting intact organelles - images typical of apoptosis. Moreover, round/ovoid electron-opaque structures appeared to be in the process of being engulfed by neighboring epithelial cells of Malassez.Conclusions: Our results demonstrate that epithelial cells of Malassez's rests undergo apoptosis in the developing and adult periodontium. Apoptosis may, together with proliferation, be part of the mechanism of turnover/remodelling of the cells of Malassez.

Role of natural killer cells in antitumor resistance

Natural killer cells constitute a population of lymphocytes able to non-specifically destroy virus-infected and some kinds of tumor cells. Since this lytic activity was shown by non-immunized animals the phenomenon is denominated natural killer (NK) activity and contrasts with specific cytotoxicity performed by cytolytic T lymphocytes (CTLs) because it does not depends on MHC-restricted peptides recognition. In fact, the main feature of most functional receptors of NK cells (NKRs) is their ability to be inhibited by different kinds of class I MHC antigens. In the middle of the 1950's, Burnet & Thomas forged the concept of tumor immunosurveillance and NK cells can be considered one of the main figures in this phenomenon both for effector and regulatory functions. In the present review the early studies on the biology of NK cells were revisited and both their antitumor activity and dependence on the activation by cytokines are discussed.

In vitro cytotoxic and non-genotoxic effects of gutta-percha solvents on mouse lymphoma cells by single cell gel (comet) assay

Chloroform and eucalyptol are widely used in clinical dentistry as gutta-percha solvents. However, these compounds may represent a hazard to human health, especially by causing injury to genetic apparatus and/or inducing cellular death. In this study, the genotoxic and cytotoxic potentials associated with exposure to chloroform and eucalyptol were assessed on mouse lymphoma cells in vitro by the single cell gel (comet) assay and trypan blue exclusion test, respectively. Both gutta-percha solvents proved to be cytotoxic at the same levels in concentrations of 2.5, 5 and 10 μL/mL (p<0.05). On the other hand, neither of the solvents induced DNA breakage. Taken together, these results suggest that although both tested compounds (chloroform and eucalyptol) are strong cytotoxicants, it seems that they are not likely to increase the level of DNA damage on mammalian cells.

Clinical outcome in chronic myeloid leukemia after allogeneic hematopoietic stem cell transplantation: The experience of the Bone Marrow Transplantation Unit of FUNFARME/BRAZIL using FISH

Investigation of the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in chronic myeloid leukemia patients is essential to predict prognosis and survival. In 20 patients treated at the Bone Marrow Transplantation Unit of São José do Rio Preto (São Paulo, Brazil), we used fluorescence in situ hybridization (FISH) to investigate the frequency of cells with BCR/ABL rearrangement at diagnosis and at distinct intervals after allo-HSCT until complete cytogenetic remission (CCR). We investigated the disease-free survival, overall survival in 3 years and transplant-related mortality rates, too. Bone marrow samples were collected at 1, 2, 3, 4, 6, 12, and 24 months after transplantation and additional intervals as necessary. Success rate of the FISH analyses was 100%. CCR was achieved in 75% of the patients, within on average of 3.9 months; 45% patients showed CCR within 60 days after HSCT. After 3 years of the allo-HSCT, overall survival rate was 60%, disease-free survival was 50% and the transplant-related mortality rate was 40%. The study demonstrated that the BCR-ABL FISH assay is useful for follow-up of chronic myeloid leukemia patients after HSCT and that the clinical outcome parameters in our patient cohort were similar to those described for other bone marrow transplantation units. ©FUNPEC-RP.