AE Cells Research Articles

Tumor cell-expressed IL-15Rα drives antagonistic effects on the progression and immune control of gastric cancer and is epigenetically regulated in EBV-positive gastric cancer.

Epstein-Barr virus associated gastric cancer (EBVaGC) often exhibits a favorable prognosis that correlates with highly methylated viral and host genes and significant immune cell infiltration compared to EBV-negative gastric cancers (GCs). Previously, it has been reported that expression of the IL-15 receptor α (IL-15Rα) is down-regulated in EBVaGC via promoter hypermethylation. In the present study, we offer a novel explanation for this puzzle by associating IL-15Rα expression with infiltration of lymphocytes in GC lesions. We investigated the expression of IL-15Rα by RT-PCR, Western-blotting and immunohistochemistry in GC cell lines and primary tissues, respectively. IL-15Rα promoter methylation was analyzed using genomic methylation sequencing. The growth behavior of GC cells was analyzed using MTT, flow cytometry, colony formation, transwell invasion and scratch wound healing assays. Demethylation of IL-15Rα was carried out using 5-Aza-CdR, and rIL-15 was added to evaluate growth promoting effects of the IL-15/IL-15Rα complex. Human peripheral blood mononuclear cells (PBMCs) were co-cultured with GC cells with/without the addition of rIL-15, after which the phosphorylation of STAT5 in PBMCs was evaluated using flow cytometry to estimate the activation of these immune cells through IL-15 binding to IL-2Rβ/γ receptors by in trans presentation. We found that EBV-positive GC cells (AE) expressed IL-15Rα at a significantly lower level than EBV-negative GC cells (AGS) due to promoter hypermethylation. In the absence of immune cells, IL-15Rα on the cancer cell surface induced a malignant phenotype, including augmented cell growth, migration and invasion, and decreased apoptosis. 5-Aza-CdR reverted AE cells to a more malignant phenotype similar to AGS cells, which may be attributed to activation of the STAT1, STAT3 and ERK1/2 pathways. However, when PBMCs were added to the GC cell cultures, these immune cells were activated as detected by increased pSTAT5 levels. Also, more GC cells underwent apoptosis. These effects were enhanced by the addition of rIL-15 and, subsequently, confirmed in EBVaGC patient samples exhibiting increased expression of T cell surface markers and activation of immune co-stimulating pathways. Our findings suggest a mechanistic explanation for the clinical association of EBVaGC with a lower IL-15Rα expression, a better prognosis and an increased lymphocyte infiltration. We propose that in highly infiltrated GCs the IL-15/IL-15Rα complex on the GC cell surface may present IL-15 in trans to IL-2Rβ/γ-expressing immune cells to activate these cells in the tumor microenvironment.

Evolution of Chikungunya virus in mosquito cells

It has been observed that replication of Chikungunya virus (CHIKV) in C6/36 Aedes albopictus cells has little effect on virus evolution. To characterize evolutionary patterns associated with CHIKV replication in mosquito cells, we performed serial passages of the LR2006 strain in Ae. albopictus cells (75 and 30 passages in C6/36 and U4.4 respectively) and Ae. aegypti cells (100 passages in AA-A20 and in AE) and studied genotypic changes accompanying adaptation during this evolutionary process. Quantitative analysis revealed cell specific patterns: low mutation rates in C6/36 cells except when a CHIKV strain pre-adapted to mammalian was used and typical features of adaptation to cell culture conditions with a high number of fixed mutations in AE and AA-A20 cells probably due to the weak permissiveness of these latter cell lines. Altogether, these results suggested that both cell line and viral strain influence rates of viral evolution. In contrast, characteristics and distribution of mutations were qualitatively very similar in all mosquito cells with a high level of parallel evolution including 4 deletion mutations. Serial passage in mammalian cells of viruses pre-adapted to mosquito cells revealed disappearance of almost all shared mutations suggesting that many of these mutational patterns are vector-specific.

Amnion Epithelial Cells of Buffalo (Bubalus bubalis) Term Placenta Expressed Embryonic Stem Cells Markers and Differentiated into Cells of Neurogenic Lineage In Vitro

ABSTRACTAim of the present study was the isolation, culture, and characterization of amniotic membrane-derived epithelial cells (AE) from term placenta collected postpartum in buffalo. We found that cultured cells were of polygonal in shape, resistance to trypsin digestion and expressed cytokeratin-18 indicating that they were of epithelial origin. These cells have negative expression of mesenchymal stem cell markers (CD29, CD44, and CD105) and positive for pluripotency marker (OCT4) genes indicated that cultured cells were not contaminated with mesenchymal stem cells. Immunofluorescence staining with pluripotent stem cell surface markers, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81 indicated that these cells may retain pluripotent stem cell characteristics even after long period of differentiation. Differentiation potential of these cells was determined by their potential to differentiate into cells of neurogenic lineages using retinoic acid. In conclusion, we demonstrate that AE cells expressed pluripotent stem cell markers and have propensity to differentiate into cells of neurogenic lineage upon directed differentiation in vitro.

Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells

BackgroundBunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.Methodology and Principal FindingsTo determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.Conclusions/SignificanceBunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.

The thrombopoietin/MPL/Bcl-xL pathway is essential for survival and self-renewal in human preleukemia induced by AML1-ETO

AbstractAML1-ETO (AE) is a fusion product of translocation (8;21) that accounts for 40% of M2 type acute myeloid leukemia (AML). In addition to its role in promoting preleukemic hematopoietic cell self-renewal, AE represses DNA repair genes, which leads to DNA damage and increased mutation frequency. Although this latter function may promote leukemogenesis, concurrent p53 activation also leads to an increased baseline apoptotic rate. It is unclear how AE expression is able to counterbalance this intrinsic apoptotic conditioning by p53 to promote survival and self-renewal. In this report, we show that Bcl-xL is up-regulated in AE cells and plays an essential role in their survival and self-renewal. Further investigation revealed that Bcl-xL expression is regulated by thrombopoietin (THPO)/MPL-signaling induced by AE expression. THPO/MPL-signaling also controls cell cycle reentry and mediates AE-induced self-renewal. Analysis of primary AML patient samples revealed a correlation between MPL and Bcl-xL expression specifically in t(8;21) blasts. Taken together, we propose that survival signaling through Bcl-xL is a critical and intrinsic component of a broader self-renewal signaling pathway downstream of AML1-ETO–induced MPL.

The Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (LARG) Is Essential for Survival and Proliferation of AML1-ETO Associated AML Cells

Abstract 923AML1-ETO (AE), the product of translocation 8;21, is the most frequently observed fusion gene in acute myeloid leukemia (AML). Although AE is not sufficient to induce leukemia by itself, it endows a significant survival and growth advantage to hematopoietic progenitors. In a search of various AML microarray databases, we found that the Leukemia-Associated Rho Guanine nucleotide exchange factor (LARG) gene expression is consistently upregulated in samples from AE-positive AML patients compared to other types of AML. This observation was confirmed in an independent cohort of AML samples by qPCR and Western Blot analysis, and in a pre-leukemia cell model generated by AE expression in CD34+ human cord blood (CB) cells compared with CB cells transduced with the MLL-AF9 (MA9) fusion gene or control retroviral vector. ChIP-PCR assays further indicate that AE directly binds to the LARG regulatory region through cis-elements containing AML1 (Runx1) binding sites. LARG, a RhoA GEF, can activate the RhoA pathway which is important for interaction between cells and their environment. We found that the AE cell lines were more adhesive to fibronectin than the MA9 cell lines. When we used lentiviral vectors expressing shRNA to suppress LARG expression in AE+ pre-leukemic cells, the cells showed a significant decrease in adhesion to fibronectin. In addition, knockdown of LARG also significantly interfered with the growth of AE cells in that the shRNA transduced cells displayed a competitive disadvantage relative to non-transduced or control-transduced cells in a proliferation assay. Cell cycle analysis revealed that LARG knockdown induced cell cycle exit, while Annexin V staining showed that LARG suppression promoted apoptosis of AE cells. Because LARG is a well-known guanine nucleotide exchange factor (GEF) for RhoA GTPase, we examined the downstream signaling events of the LARG/Rho axis, including phosphorylation of MLC and AURORA, targets of Rho-associated protein kinase (ROCK). pMLC and pAURORA were significantly down-regulated upon knockdown of LARG. Phosphorylation of Stat3, another protein downstream of RhoA signaling important for the proliferation and survival of myeloid cells, was also decreased upon LARG knockdown. These findings suggest that LARG is a transcriptional target of the AML1-ETO fusion protein and the LARG-RhoA signaling pathway plays an essential role in the proliferation and survival of AE cells. The LARG/RhoA pathway may serve as a new therapeutic target in t(8;21) AML. Disclosures:No relevant conflicts of interest to declare.

Generation of induced Pluripotent Stem Cells from Human Amnion Epithelium

Generation of induced Pluripotent Stem Cells from Human Amnion Epithelium

N-RasG12D induces features of stepwise transformation in preleukemic human umbilical cord blood cultures expressing the AML1-ETO fusion gene

AbstractAML1-ETO (AE) is a fusion product of t(8;21) observed in 40% French-American-British M2 type of acute myeloid leukemia (AML). Clinical data suggest that Ras mutation is a frequent cooperating event in t(8;21) AML. Whether constitutively active Ras promotes leukemogenesis on the t(8;21) background has not been demonstrated experimentally. Here, we retrovirally expressed N-RasG12D in AE-expressing human hematopoieticcells to investigate cooperativity. The AE/N-RasG12D cultures were cytokine-independent, enriched for CD34 positivity, and possessed increased colony-forming and replating abilities. N-RasG12D expression led to Bcl-2 up-regulation and reduced apoptosis. Ectopic Bcl-2 expression also resulted in enhanced colony-forming and replating abilities but was insufficient to sustain cytokine independence. AE/N-RasG12D cells were more sensitive to Bcl-2 inhibition with ABT-737 than parent AE cells. Enhanced engraftment of AE/N-RasG12D cells was observed on intrafemoral injection into immunodeficient mice, presumably because of improved survival in the bone marrow microenvironment. N-RasG12D promotes progression toward transfor-mation in AE-expressing cells, partially through up-regulating Bcl-2.

C-KIT mutation cooperates with full-length AML1-ETO to induce acute myeloid leukemia in mice

The full-length AML1-ETO (AE) fusion gene resulting from t(8;21)(q22;q22) in human acute myeloid leukemia (AML) is not sufficient to induce leukemia in animals, suggesting that additional mutations are required for leukemogenesis. We and others have identified activating mutations of C-KIT in nearly half of patients with t(8;21) AML. To test the hypothesis that activating C-KIT mutations cooperate with AE to cause overt AML, we generated a murine transduction and transplantation model with both mutated C-KIT and AE. To overcome the intracellular transport block of human C-KIT in murine cells, we engineered hybrid C-KIT (HyC-KIT) by fusing the extracellular and transmembrane domains of the murine c-Kit in-frame to the intracellular signaling domain of human C-KIT. We showed that tyrosine kinase domain mutants HyC-KIT N822K and D816V, as well as juxtamembrane mutants HyC-KIT 571+14 and 557-558Del, could transform murine 32D cells to cytokine-independent growth. The protein tyrosine kinase inhibitor dasatinib inhibited the proliferation of 32D cells expressing these C-KIT mutants, with potency in the low nanomolar range. In mice, HyC-KIT N822K induced a myeloproliferative disease, whereas HyC-KIT 571+14 induces both myeloproliferative disease and lymphocytic leukemia. Interestingly, coexpression of AE and HyC-KIT N822K led to fatal AML. Our data have further enriched the two-hit model that abnormalities of both transcription factor and membrane/cytosolic signaling molecule are required in AML pathogenesis. Furthermore, dasatinib prolonged lifespan of mice bearing AE and HyC-KIT N822K-coexpressing leukemic cells and exerted synergic effects while combined with cytarabine, thus providing a potential therapeutic for t(8;21) leukemia.

Equine bone marrow mesenchymal or amniotic epithelial stem cells as feeder in a model for thein vitroculture of bovine embryos

Various studies have shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In the present study we investigated the effects of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSCs) or equine amniotic epithelial stem cells (AE-SCs) on in vitro blastocysts development. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and the isolated cells were resuspended in Dulbecco Modified Earle's Medium supplemented with 10 ng/ml of basic fibroblast growth factor (bFGF). Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fragments were incubated with 0.05% trypsin for 45 min. Separated AE cells were plated in standard culture medium containing 10 ng/ml epidermal growth factor (EGF). Seven hundred and five cumulus-oocyte complexes were used and, after IVM and IVF, cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured up to day 7: (1) co-culture with cumulus cells (control); (2) co-culture with BM-MSCs; and (3) co-culture with AE-SCs. Statistical analyses were performed by ANOVA. Blastocyst developmental rates were significantly different (p < 0.001) between control, AE-SCs and BM-MSCs (respectively 35.45, 41.84 and 30.09%). In conclusion, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSCs and cumulus cells. It can be suggested that these cells secrete biologically active substances, including signalling molecules and growth factors of epithelial nature, different to those of the BM cells of mesenchymal origin.

Thrombopoietin/Bcl-Xl Signaling Is Essential for the Self-Renewal Phenotypes of AML1-ETO Expressing Human Hematopoietic Cells.

Abstract 1424Poster Board I-447AML1-ETO (AE) fusion protein is generated as a result of translocation (8;21), accounting for 40% of the FAB M2 subtype of acute myeloid leukemia (AML). This group of patients has a 5-year survival rate of approximately 50% due to disease relapse. Retroviral gene transfer of AE in CD34+ human umbilical cord blood (hUBC) cells enhances self-renewal phenotypes, making this model a powerful tool to study cellular pathophysiology in response to fusion protein expression during the early stage of disease formation. Based on the current understanding of t(8;21) AML as a stem cell disease, these AE-expressing hUBC cells may also represent the chemotherapy-non-targetable leukemic stem cells that are responsible for the relapsing disease. In the current study, we identified thrombopoietin (TPO) as an essential cytokine for the growth of AE cells. Whereas normal CD34+ hUBC cells cultured in the absence or presence of TPO showed similar growth rates, withdrawing TPO in AE cell cultures resulted in a gradual proliferative defect that was associated with loss of colony forming ability and long-term culture-initiating cells (LTC-IC) frequency. Adding TPO to methylcellulose media leads to an increase in colony numbers and an enhanced replating ability, further confirming that TPO is particularly critical for the growth of the clonogenic AE progenitor cells. Moreover, TPO withdrawal caused a decrease in the percentage of CD34+ cells, and an increase in the percentages of both CD11b+ and CD14+ cells. AE cells incubated in media without TPO adapted a more mature morphology, including nuclear segmentation and cytoplasmic granule formation. At the molecular level, various genes involved in hematopoietic cell self-renewal are downregulated in AE cells cultured without TPO. Taken together, these results indicate that TPO signaling is a critical mediator of the self-renewal phenotypes of the AE cells. Interestingly, AE cells displayed addiction to TPO-mediated signaling as early as one week after AE expression. Therefore, we further hypothesized that AE is directly involved in this process. Indeed, we found that the c-Mpl gene, which encodes the Janus Kinase 2 (JAK2)-activating TPO receptor, is upregulated in AE cells. Additionally, a chromatin immunoprecipitation assay provided evidence of direct AE binding to the promoter region of c-Mpl. These data indicate that AE directly upregulates c-Mpl gene expression in hematopoietic stem and progenitor cells. In the study of the downstream effectors of the TPO signaling, we found that Bcl-xL, an essential anti-apoptotic protein in AE cells, is maintained at high levels by TPO, mainly in a post-translational manner, suggesting that an important component of TPO signaling is to promote cell survival. In fact, recent evidence on the opposing effects of DNA damage and JAK2 activation on Bcl-xL post-translational modifications (deamidation) led us to speculate that the negative effects from increased DNA damage associated with AE expression may be counterbalanced by TPO signaling. In conclusion, our data provide a novel therapeutic approach for t(8;21) AML treatment by targeting the TPO signaling pathway, thereby augmenting conventional chemotherapy in the prevention of disease relapse. Disclosures:No relevant conflicts of interest to declare.

111 EQUINE AMNIOTIC EPITHELIAL OR BONE MARROW MESENCHYMAL STEM CELLS DIFFERENTLY SUPPORT IN VITRO EMBRYO DEVELOPMENT IN A BOVINE IN VITRO CULTURE MODEL

There are indications that the culture system and the medium composition can affect embryo quality. In fact, various studies have been shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In light of this, recently, some studies used co-culture with mouse embryonic fibroblasts in the effort to improve the development of bovine and ovine in vitro-derived embryos. Despite the progress in equine IVM and ICSI technologies and the different culture conditions reported for preimplantation development of ICSI fertilized horse oocytes, the yield of blastocysts remained low. In the present study we investigated the benefits of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSC) or equine amniotic epithelial stem cells (AE-SC) on blastocyst development. This study employed the bovine embryo as a model and represents the initial step towards standardization of a protocol for the culture of equine embryos in our laboratory. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and layered over Hystopaque™ 1.080, then centrifuged for 20 min at 400g and 4°C. Cell pellets were resuspended in 10 mL Dulbecco Modified Earle’s Medium supplemented with 10% fetal calf serum, 1% non-essential amino acids, penicillin (100 U mL–1) and streptomycin (100 μg mL–1) and seeded in 24-well plates. Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fractions were incubated at 37°C with 0.05% trypsin for 45 min. Separated AE cells were plated on 25 cm2 flask in standard culture media containing 10 ng mL–1 epidermal growth factor. Seven hundred fifty cumulus–oocyte complexes with a homogeneous cytoplasm and two or more layers of cumulus cells were used. After IVM and IVF cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured for up to Day 7: 1) co-culture with granulosa cells (control); 2) co-culture with BM-MSC; 3) co-culture with AE-SC. The culture medium was TCM 199 + 10% fetal bovine serum, pyruvate and gentamicin at 38.5°C in 5% CO2. Statistical analyses was performed by chi square test. Blastocysts developmental rates were similar among control, AE-SC and BM-MSC (35%, 41% and 30%, respectively), but the co-culture with AE-SC gave a significantly greater percentage of blastocysts compared to BM-MSC (P &lt; 0.05). In conclusion, despite the absence of a significant increment in blastocysts attainment using stem cells as feeders for embryo culture, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSC. It can be suggested that these cells secrete biologically active substances including signaling molecules and growth factors of epithelial nature different from those of the BM cells of mesenchymal origin. Regione Lombardia is acknowledged for the “Dote Ricercatori” fellowship to V.M.

Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability.

The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.

Telomerase Promotes the Self-Renewal of Human Hematopoietic Progenitor Cells but Does Not Maintain Telomere Length.

Telomerase is a reverse transcriptase that adds telomeric sequences onto chromosome ends. During hematopoiesis, telomerase activity is high in proliferating hematopoietic stem and progenitor cells, but decreases sharply as cells differentiate. Approximately 90% of all human cancers are telomerase positive, including samples from AML patients. To understand the role of telomerase in human hematopoietic stem and progenitor cell proliferation and differentiation, and in the development of leukemia, we established stable long-term cultures expressing high levels of telomerase by retrovirus-mediated transduction of the h-TERT (catalytic subunit of telomerase) gene into AML1-ETO-expressing CD34+ (AE) cells. We have previously shown that these AE cells maintain CD34 expression and proliferate for 7 months in culture but gradually lose telomere length and are not immortal, providing a good model for studying the molecular mechanisms involved in telomere shortening. Strikingly, telomerase expression enhanced the self-renewal ability of the human hematopoietic progenitor cells, as shown by continuous replating ability in methylcellulose assays. We also detected a pronounced delay in the differentiation of the progenitor cells upon telomerase expression, and an expansion of the progenitor pool. Telomerase expression promoted proliferation due to increased cell cycle progression as well as a survival advantage. At the same time, these cells demonstrated a progressive decline in telomere length, despite telomerase activity equivalent to that detected in leukemia cell lines. We conclude that expression of hTERT expands the pool of hematopoietic progenitors but does not maintain long telomeres in human CD34+ cells. hTERT plays a critical role not only in telomere homeostasis for genetic stability but also in cellular proliferation, differentiation, and self-renewal, functions that may be part of hTERT involvement in leukemogenesis. These data open an important debate regarding the specific contribution of telomerase expression to the leukemic phenotype and the potential timing of this essential hit in the progression of the disease.

Targeting fusion protein/corepressor contact restores differentiation response in leukemia cells

The AML1/ETO and PML/RARalpha leukemia fusion proteins induce acute myeloid leukemia by acting as transcriptional repressors. They interact with corepressors, such as N-CoR and SMRT, that recruit a multiprotein complex containing histone deacetylases on crucial myeloid differentiation genes. This leads to gene repression contributing to generate a differentiation block. We expressed in leukemia cells containing PML/RARalpha and AML1/ETO N-CoR protein fragments derived from fusion protein/corepressor interaction surfaces. This blocks N-CoR/SMRT binding by these fusion proteins, and disrupts the repressor protein complex. In consequence, the expression of genes repressed by these fusion proteins increases and differentiation response to vitamin D3 and retinoic acid is restored in previously resistant cells. The alteration of PML/RARalpha-N-CoR/SMRT connections triggers proteasomal degradation of the fusion protein. The N-CoR fragments are biologically effective also when directly transduced by virtue of a protein transduction domain. Our data indicate that fusion protein activity is permanently required to maintain the leukemia phenotype and show the route to developing a novel therapeutic approach for leukemia, based on its molecular pathogenesis.

Hemoglobin E: a balanced polymorphism protective against high parasitemias and thus severe P falciparum malaria

Hemoglobin E is very common in parts of Southeast Asia. The possible malaria protective effects of this and other inherited hemoglobin abnormalities prevalent in Thailand were assessed in a mixed erythrocyte invasion assay. In vitro, starting at 1% parasitemia, Plasmodium falciparum preferentially invaded normal (HbAA) compared to abnormal hemoglobin (HbH, AE, EE, HCS, beta-thalassemia E) red cells (HRBCs). The median (range) ratio of parasitization of HRBCs (n = 109) compared to the controls of different major blood groups was 0.40 (0.08, 0.98), less than half that of the normal red cells (NRBCs) compared to their controls 0.88 (0.53, 1.4; P =.001). The median (range) parasitemia in the HRBCs was 2% (0.1%-9%) compared to 5.2% (1.2%-16.3%) in the NRBCs (P =.001). The proportion of the RBC population that is susceptible to malaria parasite invasion can be described by a selectivity index (SI; observed number of multiply invaded RBCs/number predicted). The heterozygote AE cells differed markedly from all the other cells tested with invasion restricted to approximately 25% of the RBCs; the median (range) SI was 3.8 (1-15) compared with 0.75 (0.1-0.9) for EE RBCs (P <.01). Despite their microcytosis, AE cells are functionally relatively normal in contrast to the RBCs from the other hemoglobinopathies studied. These findings suggest that HbAE erythrocytes have an unidentified membrane abnormality that renders the majority of the RBC population relatively resistant to invasion by P falciparum. This would not protect from uncomplicated malaria infections but would prevent the development of heavy parasite burdens and is consistent with the "Haldane" hypothesis of heterozygote protection against severe malaria for hemoglobin E.

Partial Characterization of a Human ZincDeficiency Syndrome by Differential Display

The effect of the acrodermatitis enteropathica mutation (AE) on gene expression was investigated using differential display. Two differentially expressed cDNAs were partially characterized. The NA8 cDNA (HT11A anchor and HAP 8 random primer pair) was expressed in greater quantity in normal fibroblasts, was 249 bp, and hybridized to three mRNA species (2 kb, 1 kb, 0.8 kb). Northern blot analysis indicated that the relative amounts of the AE mRNA species were reduced by 73%, 75%, and 52%, respectively. The cDNA sequence exhibited 92-93% homology to the human cytochrome oxidase subunit II, as analyzed through the GenBank database. The AEG4 cDNA species (HT11G anchor and HAP 4 random, primer pair) was expressed in greater quantity in AE fibroblasts, was 197 bp, and hybridized to two mRNA species (9 kb, 4 kb). Northern blot analysis indicated that the 9-kb mRNA species was present equally in AE and normal cells, but the 4-kb mRNA species was only present in the AE fibroblasts. The cDNA sequence exhibited 92% homology to LINE1 human retrotransposons, as analyzed through the GenBank database. The functional relationship between the mutation and the reduced expression of cytochrome oxidase subunit II is unknown at this time and needs to be addressed. The increased expression of the LINE1 element in AE fibroblasts may be indicative of an insertion mutation affecting the mRNA of a protein involved in zinc transport, a prospect which requires further investigation.

Glucose uptake rate as a tool to estimate hybridoma growth in a packed bed bioreactor

The immobilisation of cells in a perfusion cul- ture allows to obtain a high cell concentration and an ef- ficient removal of the catabolites without cell loss. A disadvantage of this system is that the cell density cannot be directly monitored. The cellular metabolism is just followed by online measurements of pH and dissolved oxygen (DO) and off-line determinations of residual me- tabolites. In this article, we report a high cell density achieved by the cultivation of a hybridoma in a bubble- column bioreactor filled with hollow glass cylinders. The parameters monitored during the cultivation were pH, temperature, DO, glucose, lactate and monoclonal anti- body. The glucose uptake rate was used to estimate the cell concentration along the time. The maximum cell concen- tration calculated for the considered cultivation time was 2.7 · 10 7 cell AE ml )1 . The glucose concentration in the media decreased stepwise twice, causing a decrease on the specific growth rate, while maintaining high antibody productivity levels. Maximum monoclonal antibody pro- ductivity was 503 lg AE l )1 AE day )1 and specific productivi- ty, considering calculated cell density, was 0.019 ng AE cell )1 AE day )1 .

Precise excision of TTAA‐specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera

Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short-inverted-repeat elements are characterized by their extreme specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggyBac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN-368 cells or IPLB-SF21 AE cells. The precise excision of these elements in infected IPLB-SF21 AE cells occurs in the absence of either tagalong or piggyBac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion in a single family of unique transposons.

A functional hepatitis B virus X protein produced in insect cells

We have constructed a recombinant baculovirus from Autographa californica nuclear polyhedrosis virus, called AcX, that expresses the gene encoding the hepatitis B virus X protein in infected Spodoptera frugiperda (Sf21AE) insect cells. A 16.5-kDa monomer and a 33-kDa dimer of the X protein were detected in extracts from AcX-infected cells on immunoblots using a polyclonal anti-X antibody. The biological activity of the insect cell-produced X protein was assayed by fusing AcX-infected Sf21 AE cells with African green monkey kidney (CV-1) cells and then transfecting the fused cells with the reporter plasmid pSV2cat. The expression of the cat gene in CV- 1:Sf21AE(AcX) fusions was seven times higher than that derived from CV-1:Sf21AE(AcMNPV) fusions, indicating that the insect cell-produced X protein was functional. The transactivation function of the insect cell-produced X protein was also verified by scrape-loading nuclear extracts of AcX-infected Sf21AE cells into pSV2cat-transfected CV-1 cells. Treatment of the AcX-infected cell nuclear extracts with anti-X antisera prior to scrape-loading eliminated the transactivating activity of the extracts. We conclude that the insect cell-produced X protein was functionally identical to that generated in mammalian cells.