Cell Line KG1a Research Articles

Indirubin-3′-monoxime inhibits autophosphorylation of FGFR1 and stimulates ERK1/2 activity via p38 MAPK

Indirubin-3'-monoxime is a derivative of the bis-indole alkaloid indirubin, an active ingredient of a traditional Chinese medical preparation that exhibits anti-inflammatory and anti-leukemic activities. Indirubin-3'-monoxime is mainly recognized as an inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3. It inhibits proliferation of cultured cells, mainly through arresting the cells in the G1/S or G2/M phase of the cell cycle. Here, we report that indirubin-3'-monoxime is able to inhibit proliferation of NIH/3T3 cells by specifically inhibiting autophosphorylation of fibroblast growth factor receptor 1 (FGFR1), blocking in this way the receptor-mediated cell signaling. Indirubin-3'-monoxime inhibits the activity of FGFR1 at a concentration lower than that required for inhibition of phosphorylation of CDK2 and retinoblastoma protein and cell proliferation stimulated by fetal calf serum. The ability of indirubin-3'-monoxime to inhibit FGFR1 signaling was similar to that of the FGFR1 inhibitor SU5402. In addition, we found that indirubin-3'-monoxime activates long-term p38 mitogen-activated protein kinase activity, which stimulates extracellular signal-regulated kinase 1/2 in a way unrelated to the activity of FGFR1. Furthermore, we show that indirubin-3'-monoxime can inhibit proliferation of the myeloid leukemia cell line KG-1a through inhibition of the activity of the FGFR1 tyrosine kinase. The data presented here demonstrate previously unknown activities of indirubin-3'-monoxime that may have clinical implications.

Differential effects of bexarotene on intrinsic and extrinsic pathways in TRAIL-induced apoptosis in two myeloid leukemia cell lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death (apoptosis) preferentially in tumor cells. However, not all cancer cells are sensitive to TRAIL. We determined whether ligation of the retinoid receptor, RXR, would sensitize cells to TRAIL-mediated apoptosis. The leukemic cell lines KG1a (apoptosis-resistant) and ML-1 (apoptosis-sensitive) were treated with the RXR-specific retinoid bexarotene, TRAIL, or both, and apoptosis was determined. In KG1a cells, bexarotene downregulated FLIPLong and activated caspase-8, thereby allowing for TRAIL-triggered apoptosis. Overexpression of FLIPLong in ML-1 cells abrogated apoptosis. In unmodified ML-1 cells bexarotene enhanced programmed cell death via truncation of Bid and release of cytochrome C. Blockade of caspase-8 prevented enhancement in both cell lines; blockade of caspase-9 had a significant effect only in ML-1 cells. Thus, the effect of bexarotene on TRAIL-mediated programmed cell death involved proximal events of the extrinsic pathway; however, downstream signals involved the intrinsic pathway in ML-1 but not in KG1a cells. These studies add further information to the regulation of programmed cell death in leukemic cells that have to be considered when designing therapeutic strategies.

HCELL Is the Major E- and L-Selectin Ligand Expressed on Human Hematopoietic Progenitor Cells and Colon Carcinoma Cells.

Selectins are a class of cell surface proteins specialized to mediate adhesive interactions under hydrodynamic shear flow conditions, and are critical in the trafficking of hematopoietic progenitor cells (HPC) into the bone marrow and dissemination of cancer cells in hematogenous metastasis, among other biological events. More specifically, E-selectin expressed by endothelial cells has been shown to mediate shear-resistant tethering/rolling of cells recruited from the fluid stream, while L-selectin expressed by distinct leukocyte subsets has been variously shown to mediate lymph node homing, as well as the formation of heterotypic cell aggregates in bulk flow. We have previously reported that the primitive CD34+ human hematopoietic progenitor cell (HPC) line KG1a exhibits stronger E-/L-selectin ligand activities than HL60, a more differentiated CD34− human hematopoietic cell line. These findings were attributed to the HPC/KG1a restricted expression of the sialofucosylated CD44 glycoform termed HCELL (Hematopoietic Cell E-/L-selectin Ligand), since both cell lines express approximately equivalent levels of CD44 and the well-characterized selectin ligand P-selectin glycoprotein ligand-1 (PSGL-1). To directly assess whether HCELL functions as the high affinity E-/L-selectin ligand on KG1a cells, HCELL expression was silenced using lentiviral siRNA targeting CD44, and the E-/L-selectin ligand activities were subsequently compared between untreated and transduced KG1a cells. CD44 siRNA transduction of KG1a cells decreased CD44 (i.e. HCELL) expression levels >90% mean fluorescence intensity (MFI) relative to untreated and vector alone-transduced cells as measured by flow cytometry. CD44 targeting was specific, as expression of PSGL-1 was not reduced upon CD44 siRNA transduction, nor were CD29, CD49d, or CD49e levels affected. Functionally, at venous shear stress levels (≤4 dyn/cm2), CD44 siRNA-transduced KG1a cells rolled markedly faster than untreated and vector alone-transduced cells on a monolayer of E-selectin transfected Chinese hamster ovary (CHO-E) cells. At arterial and pathological shear (>4 dyn/cm2), control cells maintained rolling interactions on CHO-E cells, whereas CD44 siRNA-transduced cells readily detached from the monolayer. Comparable results were obtained for L-selectin-dependent peripheral blood mononuclear cell rolling on CD44 siRNA-transduced versus untreated and vector alone-transduced KG1a cells. Furthermore, the HCELL-negative HL60 cells supported similarly weak binding of E- and L-selectin as CD44 siRNA-transduced KG1a cells. Collectively, the data directly show that HCELL is the predominant ligand that stabilizes shear-resistant HPC binding to E- and L-selectin. We have also previously reported that the human colon carcinoma cell line LS174T expresses HCELL. To examine whether HCELL on LS174T cells functions as a selectin ligand, we again suppressed expression through targeted gene silencing by lentiviral CD44 siRNA transduction. Similar to KG1a cells, specific CD44/HCELL silencing on LS174T cells was achieved (>90% reduction in MFI relative to untreated and vector controls), resulting in significant inhibition of E- and L-selectin binding capabilities at physiologically relevant shear stress levels. In summary, these findings demonstrate that HCELL is the most potent E-/L-selectin ligand mediating shear-resistant adhesive interactions on human cells.

Targeting of Heat Shock Protein 32 (Hsp32) in Neoplastic Cells by Styrene Maleic Acid Zinc Protoporphyrin (SMA-ZnPP) Is Associated with Reduced Growth and Induction of Apoptosis.

Heat shock protein 32 (Hsp32), also known as heme oxygenase-1 (HO-1), is a stress-related survival factor that has recently been implicated in enhanced survival of neoplastic cells. We here show that Hsp32/HO-1 is expressed abundantly in primary neoplastic cells in various solid tumors and hematopoietic neoplasms such as acute myeloid leukemia (AML) or chronic myeloid leukemia (CML), and in respective cell lines including the AML cell lines HL60, KG1, KG1a, and U937, CML cell lines K562 and KU812, eosinophilic leukemia cell line EOL-1, mast cell leukemia cell line HMC-1, myeloma cell lines RPMI8226 and U266, breast cancer cell line MDA MB 231, lung cancer cell line A549, pancreatic carcinoma cell line BxPC-3, colon carcinoma cell lines COLO201, COLO205, COLO320DM, and DLD-1, and the ovarian carcinoma cell line OVCAR-3. Expression of Hsp32 mRNA was demonstrable by RT-PCR and Northern blotting, and expression of the Hsp32 protein by Western blotting and immunocytochemistry. The CML-specific oncoprotein BCR/ABL and the transforming oncoprotein KIT D816V that is expressed in neoplastic mast cells, were found to promote expression of Hsp32 in Ba/F3 cells. In order to examine the functional role of Hsp32 in neoplastic cells, a specific siRNA was employed. Expression of Hsp32 siRNA resulted in reduced viability and induction of apoptosis in all cell lines tested. To further explore the value of Hsp32 as a target in neoplastic cells, a novel specific Hsp32-targeting compound, styrene maleic acid copolymer zinc protoporphyrin micelles (SMA-ZnPP) was applied. Exposure to SMA-ZnPP resulted in a significant decrease in proliferation determined by 3H-thymidine uptake, in all cell lines as well as in all primary neoplastic cells tested (AML, n=5; CML, n=5; mastocytosis, n=3; breast cancer, n=2; lung cancer, n=1). As assessed by AnnexinV-staining, Tunel assay and electron microscopy, the growth-inhibitory effects of SMA-ZnPP were found to be associated with induction of apoptosis. In each cell type, the effect of SMA-ZnPP on growth was dose-dependent and found to occur at pharmacologic concentrations (IC50 1–30 μM). Moreover, SMA-ZnPP was found to synergize with various anti-neoplastic drugs (tumor cell lines: cisplatin, leukemias: cytarabine, myeloma: bortezomib) in producing growth inhibition. In summary, these data show that Hsp32/HO-1 is an important survival factor and novel interesting target in various hematopoietic and non-hematopoietic neoplasms. Based on these data, it seems desirable to explore the value of the Hsp32-targeting drug SMA-ZnPP in clinical trials in patients with leukemias and solid tumors.

Generation of Highly Efficient Recombinant Adeno-Associated Virus (rAAV) Vectors Targeted to Chronic Myelogenous Leukemia (CML) - and CD34+ Progenitor Cells Obtained by the „AAV Random Peptide Library“ Method.

Resistance of chronic myelogenous leukemia (CML) to imatinib is a serious clinical challenge. In vivo targeted gene therapy may be a strategy to overcome resistance. For gene therapy of CML standard rAAV2-based vectors lack the required gene transfer efficiency as well as the in vivo selectivity. An advancement in vector development (Muller et al., Nat. Biotech., 2003) now allows the generation of rAAV capsid mutants that offer higher target cell specificity and efficiency. We used the “AAV random peptide library” method to generate capsid mutants efficient and specific for CML cell lines, primary CML cells and primary human peripheral blood progenitor cells PBPC. After previously applying this method successfully on CML cell lines, an effective vector for CML cell lines could be obtained. On primary human CML and PBPC the new vector was more efficient than rAAV2 but still at an unsatisfying level. To increase the gene transfer on primary cells we applied the “AAV library” on primary human CML and PBPC. A total of 44 different mutant capsid clones were obtained, most of them at three or less copies. Four clones were observed >10x to 39x and were selected for further testing. To determine efficiency and specificity, a panel of leukemic (CML, imatinib-sensitive and -resistant, and AML) and other solid tumor cell lines were transduced (moi 100). On a panel of leukemic cell lines similar or higher gene transfer efficiency of the rAAV capsid mutants was observed compared to standard rAAV2 vectors. The higher transduction efficiency in imatinib-resistant cell line LAMA84-R than in their sensitive counterpart LAMA84-S was particularly striking, as the difference in susceptibility for the capsid mutants vs rAAV2 in LAMA84-S (table 1). On solid tumor cell lines rAAV2 was more efficient, confirming the higher specificity of our capsid mutants. Compared to our previous work (<1% GFP+ cells), the new capsid mutants now result in efficient gene transfer into the AML cell line KG1a, and to lesser extend the CML cell line EM3. For both cell lines gene transfer with the capsid mutants was readily more efficient than with standard rAAV2 vectors. Preliminary data on primary PBPC showed a uniformly higher transduction efficiency for all capsid mutants (%GFP_mut1-4: 13.6% ± 2.2% vs %GFP_AAV2: 5.3%). Experiments with primary human CML cells are ongoing. Using the “AAV random peptide library” method on primary cells, we were able to generate rAAV vectors with higher transduction efficiency on leukemic cell lines and primary PBPC than standard rAAV2 vectors. The development of these highly efficient AAV mutant capsid vectors holds promise for future development.Gene transfer efficiency (%GFP+ cells)Cell lineCapsid mutantStandard rAAV2LAMA84-S78.2 ± 7.99.2 ± 2.5LAMA84-R97.8 ± 0.393.0 ± 0.2HL-6054.0 ± 5.457.6 ± 8.2BV17348.4 ± 7.430.6 ± 1.8EM33.9 ± 0.70.2 ± 0.2KG1a24.7 ± 0.712.8 ± 5.0Data are given as mean ± SD

Reversal of p15/ INK4b hypermethylation in AML1/ETO-positive and -negative myeloid leukemia cell lines

In vitro and in vivo, myeloid leukemic and preleukemic cells exhibit variable sensitivity to the antiproliferative and proapoptotic effects induced already at low concentrations of DNA methyltransferase (DNMT) inhibitors. The molecular mechanisms underlying this variable sensitivity of leukemic blasts to azanucleosides such as 5-azacytidine and 5-aza-2′-deoxycytidine (DAC) may involve modifier effects of specific fusion proteins such as AML1/ETO. The cyclin-dependent kinase inhibitor p15/ INK4b is one potential target of DNA demethylating activity in AML and MDS where it is frequently silenced by hypermethylation. To study sensitivity to DAC in myeloid leukemia cells, we chose the myeloid cell lines Kasumi-1 (expressing AML1/ETO), KG-1 and KG-1a (both AML1/ETO-negative) all of which a highly methylated p15/ INK4b gene. Treatment with DAC resulted in dose-dependent regional demethylation of p15/ INK4b in Kasumi-1 and KG-1, but only to a modest degree in KG-1a cells. Demethylation was associated with induction of p15/INK4b protein expression. Growth-inhibitory and proapoptotic activity of DAC was significantly higher in Kasumi-1 than in KG-1a cells, and sensitization of cells to a cooperating effect of All-trans retinoic acid and of the histone deacetylase (HDAC) inhibitor Trichostatin A was observed. DAC-induced growth inhibition and apoptosis were enhanced when AML1/ETO was conditionally expressed in AML1/ETO-negative U-937 cells. In conclusion, hypomethylation and reactivation of p15/ INK4b in myeloid cell lines are among the molecular events associated with DAC-induced growth arrest and apoptosis. Further studies of AML1/ETO as a modifier of the epigenotype and sensitivity of myeloid cells to inhibitors of DNMTs and HDACs appear warranted.

Effects of Na +/H + exchanger inhibitors on subcellular localisation of endocytic organelles and intracellular dynamics of protein transduction domains HIV–TAT peptide and octaarginine

Protein transduction domains such as those derived from the HIV protein TAT have great potential as vectors for delivery of therapeutic entities such as genes and proteins into cells. Extensive studies have shown that a major fraction of the most studied variants enters cells via an endocytic mechanism. However, controversy surrounds the exact uptake mechanism and whether a specific pathway is utilised. Studies showing inhibition of uptake of protein transduction domains in the presence of ion-transport inhibitors such as amiloride and its more potent analogue 5-( N-ethyl- N-isopropyl) amiloride (EIPA) suggest a link between peptide internalisation and macropinocytosis. In this study, using immunolabelling of early and late components of the endocytic pathway, we show that treatment of cells with EIPA and to a lesser extent amiloride affects the morphology and subcellular location of early, late endosomes and lysosomes. Enlarged early and late endocytic structures were observed in EIPA-treated cells, and these organelles accumulated in a perinuclear region. Results from experiments investigating the effects of EIPA on distribution of fluorescent octaarginine were in agreement with the immunolocalisation studies. Treatment of the CD34 + leukaemia cell line KG1a with EIPA in the presence of fluorescent conjugates of HIV–TAT peptide and octaarginine showed distinct vesicular staining in agreement with untreated cells but EIPA-treated cells were additionally characterized by increased localization of the peptides in the cytosol. At levels previously shown to inhibit uptake of HIV–TAT peptide and octaarginine in other cell lines, EIPA was without major effect on uptake of both peptides in KG1a cells.

Preclinical Assessment ofFHITGene Replacement Therapy in Human Leukemia Using a Chimeric Adenovirus, Ad5/F35

Expression of the FHIT protein is lost or reduced in most solid tumors and a significant fraction of hematopoietic malignancies. Adenovirus 5 (Ad5) virus or adeno-associated viral vectors have been used to study the tumor suppressor function of FHIT in solid tumors, but these tools have not been effective in leukemias. We have generated a chimeric FHIT-containing adenovirus composed of Ad5 and the group B adenovirus called F35 with which we have been able to efficiently infect hematopoietic cells. Infection efficiency of Ad5/F35-FHIT and Ad5/F35-GFP viruses was tested in leukemia cell lines that lacked FHIT expression, and biological effects of successful infection were assessed. An acute myelogenous leukemia, a chronic myelogenous leukemia, and four acute lymphoblastic leukemia human cell lines were examined as well as two EBV-transformed B lymphoblastoid cell lines that expressed endogenous FHIT. Two of four acute lymphoblastic leukemia cell lines, Jurkat and MV4;11, which were efficiently infected with Ad5/F35-FHIT, underwent growth suppression and massive induction of apoptosis without apparent activation of caspase-8 or caspase-2 and late activation of caspase-3. Treatment of infected cells with caspase-9 and caspase-3 inhibitors partially blocked FHIT-induced apoptosis. The two remaining infected acute lymphoblastic leukemia cell lines, Molt-3 and RS4;11, were apparently unaffected. Restoration of FHIT expression in the chronic myelogenous leukemia K562 cell line and the acute myelogenous leukemia KG1a cell line also induced apoptosis but at later time points than seen in the acute lymphoblastic leukemia Jurkat and MV4;11 cell lines. I.v. injection of Ad5/F35-FHIT-infected Jurkat cells resulted in abrogation of tumorigenicity in the NOD/SCID xenogeneic engraftment model. FHIT restoration in some FHIT-deficient leukemia cells induces both antiproliferative and proapoptotic effects involving the intrinsic caspase apoptotic pathway.

In vitro anti-cancer activity of a novel microbial fermentation product on human carcinomas

The possible anti-proliferation and cell death induction potential of a novel microbial fermentation extract named as oncogen XP-180 (or simply as XP-180) was tested on three human solid tumour carcinoma cell lines (non-small cell lung cancer A549, breast cancer MDA-MB231, liver adenocarcinoma SK-Hep1) and on the acute myelogenous leukaemia KG1a cell line. Anti-proliferative activity of XP-180 was observed on all of these cancer cell lines with comparable efficiency and in a dose-dependent manner. Morphological investigation further suggested that common features of apoptosis, including cell shrinkage and rounding, are present in XP-180 treated cells. Loss of adhesion properties of these solid tumour cell lines was observed upon XP-180 incubation. Anchorage-dependent clonogenicity assay on solid tumour cell lines and semi-solid methylcellulose colony formation assay on leukaemia cell line further revealed that XP-180 strongly inhibited the regeneration potential of these cancer cells. Using KG1a as an experimental model system, XP-180 was shown to stimulate the activity of caspase 3, 8 and 9 without significant change in caspase 6 activity. Furthermore, XP-180 readily induced collapse of mitochondrial membrane potential after 2 h of incubation. However, the use of the generic caspase specific inhibitor Z-VAD-FMK does not significantly reverse XP-180 mediated cell death. The results obtained suggest that XP-180-mediated cancer cell death could involve mitochondria and both caspase-dependent and -independent pathways. Therefore, XP-180 is an efficient anti-cancer regimen in vitro.

Uptake of functionalized, fluorescent-labeled polymeric particles in different cell lines and stem cells

Labeling of cells with particles for in-vivo detection is interesting for various biomedical applications. The objective of this study was to evaluate the feasibility and efficiency labeling of cells with polymeric particles without the use of transfection agents. We hypothesized that surface charge would influence cellular uptake. The submicron particles were synthesized by the miniemulsion process. A fluorescent dye which served as reporter was embedded in these particles. The surface charge was varied by adjusting the amount of copolymerized monomer with amino group thus enabling to study the cellular uptake in correlation to the surface charge. Fluorescent-activated cell sorter (FACS) measurements were performed for detecting the uptake of the particles or attachment of particles in mesenchymal stem cells (MSC), and the three cell lines HeLa, Jurkat, and KG1a. These cell lines were chosen as they can serve as models for clinically interesting cellular targets. For these cell lines—with the exception of MSCs—a clear correlation of surface charge and fluorescence intensity could be shown. For an efficient uptake of the submicron particles, no transfection agents were needed. Confocal laser scanning microscopy and transmission electron microscopy (TEM) revealed differences in subcellular localization of the particles. In MSCs and HeLa particles were mostly located inside of cellular compartments resembling endosomes, while in Jurkat and KG1a, nanoparticles were predominantly located in clusters on the cell surface. Scanning electron microscopy showed microvilli to be involved in this process.

Intracellular Traffic and Fate of Protein Transduction Domains HIV-1 TAT Peptide and Octaarginine. Implications for Their Utilization as Drug Delivery Vectors

Transduction domains such as those derived from the HIV-TAT protein are candidate vectors for intracellular delivery of therapeutic macromolecules such as DNA and proteins. The mechanism by which they enter cells is controversial, and very little spatial information regarding the downstream fate of these peptides from the plasma membrane is available. We studied endocytic traffic of fluorescent conjugates of HIV-TAT peptide and octaarginine in human hematopoietic cell lines K562 (CD34-) and KG1a (CD34+) and substantiated our findings in epithelia cells. Both peptides were efficiently internalized to endocytic pathways of both hematopoietic cell lines; however, comparative analysis of the intracellular location of the peptides with endocytic probes revealed major differences in spatial organization of their endocytic organelles and their interaction with the peptides at low temperatures. Double labeling confocal microscopy demonstrates that prelabeled lysosomes of all the tested cells are accessible to internalized peptides within 60 min of endocytic uptake. Incubation of cells with nocodazole and cytochalasin D inhibited peptide traffic from early to late endosomal structures, demonstrating a cytoskeletal requirement for lysosomal delivery. Disruption of Golgi and endoplasmic reticulum dynamics was without effect on peptide localization, suggesting that endosomes and lysosomes rather than these organelles are the major acceptor compartments for these molecules.

Complex Regulation of the Homeobox Transcription Factor TGIF: Differential Expression of Multiple 5′ Isoforms.

TG-interacting factor (TGIF) is a transcriptional repressor belonging to the TALE (three amino acid loop extension) class of homeobox proteins. In addition to its involvement by mutation or deletion in the inherited craniofacial disorder holoprosencephaly, we have shown that expression of TGIF is highly predictive of relapse and survival in acute myelogenous leukemia (AML). To better understand how TGIF expression is regulated, we characterized its genomic structure using expressed sequence tag analysis, reverse transcriptase-coupled PCR, and rapid amplification of cDNA ends. These studies revealed a complex pattern of gene expression, with 15 splice isoforms and 11 alternative 5′ exons spread over 40 kb of DNA, suggesting the presence of multiple promoters. Real-time and semi-quantitative PCR analysis showed these isoforms were differentially expressed in various adult tissues, leukemic cell lines, and AML blasts. Further, isoform C was found to be the major RNA product in hematopoietic cells, contributing significantly to total TGIF expression in leukemic cell lines TF1a, U937, AML-193, KG-1a, Kasumi-1, K562, GDM-1, HL-60 and AML blasts. This analysis suggests that altered splicing and/or expression of specific isoforms could be responsible for the reduced levels of TGIF message observed in AML blasts and cell lines. The unusually complex structure of the TGIF gene may enable its precise regulation during normal hematopoiesis and may be relevant to its reduced expression in myeloid leukemias.

Reactivity of a Novel Antibody ZCH-2B8a on Normal and Malignant Hematopoietic Cells.

Zhejiang Children's Hospital (ZCH)-2B8a antibody was recently generated in our laboratory using leukemia cell line KG1a as immunogen. This antibody was submitted to the 8th International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA8) and the results showed that the antibody recognized an unknown molecule on the surface of some blood cells. In this study, the reactivity of the antibody on normal and malignant hematopoietic cells was investigated and its diagnostic and therapeutic significances were explored. Materials and Methods 3 specimens each of normal peripheral blood, bone marrow/G-CSF mobilized peripheral blood CD34+ stem/progenitor cells (PBSCs), 13 hematopoietic cell lines and the cells from 100 leukemia patients (male 58: female 42, 33 cases of children and 67 adults, aged 11 month ~ 77 yrs) were enrolled into this study. A multi-parameter flow cytometry with CD45 gating strategy was used to analyze the cells. Results 2B8a weakly reacted with a few B lymphocytes (34.3±15.91%), monocytes(44.4±37.77%), granulocytes (26.4±22.40%), but not reacted with other blood cells such as T cells (0.6±0.37%), NK cells (2.6±1.56%), DC cells (8.4±8.18%), erythrocytes (0.4±0.21%) and platelets (0.3±0.28%). It was reactive with 38.0±9.09% of bone marrow CD34+ cells with 21.0±22.00% of CD38+ and 25.3±22.6% of CD38- subsets (P = 0.5). In G-CSF mobilized PBSCs, 2B8a recognized 11.7±18.73% of CD34+ PBSCs with 9.7±15.20% of CD38+ and 33.3±57.74% of CD38- subsets. The positive rates of 2B8a on a panel of hematopoietic cell lines were shown in Table 1. The fresh leukemia cell analysis showed that the antibody primarily reacted with lymphoid leukemias, of which acute lymphocytic leukemia (ALL) and chronic lymphocytic leukemia (CLL) accounted for 29%(9/31) and 40%(2/5), respectively. It did not react with most cases of myeloid leukemias, with only 5%(3/57) of acute myeloid leukemia(AML) and 0% (0/6) of chronic myelogeneous leukemia (CML) cases, in which significant difference between myeloid and lymphoid leukemias (χ2= 10.29, P < 0.01) was found; In ALL, 2B8a only reacted with B lineage (32%, 9/28), while not with T and NK lineage leukemias. In B lineage leukemia, early Pre-B ALL was the most reactive type of disease, positive in 60% of cases, significantly higher than cALL cases (5/21, 24%, P = 0.0173) with declining expression pattern with cell maturation. There was no reactivity in mature B-ALL but it was strongly reactive with lymphoma cells suggesting the possibility for this antibody as a potential targeting therapy for patients with B lineage lymphoma. Conclusions 2B8a may be used in distinguishing lymphoid from myeloid leukemia. It might hold a potential application for targeting therapy for patients with leukemia and malignant lymphoma. (This work was supported by the grant (No. 398427) from Zhejiang Provincial Scientific Fundation).Table 1Reactivity of 2B8a on hematopoietic cell linesCell linesSpecificityPositivity ±%CEMT cell leukemia5.0±0.67JMT cell leukemia5.1±0.84Molt-3T cell leukemia8.3±3.59Molt-4T cell leukemia4.3±3.29Nalm-6B cell leukemia94.9±0.78RajiB cell lymphoma98.8±0.22SMS-SBB cell lymphoma95.6±2.17KG1aMyeloblastic leukemia16.7±1.93U937Monoblastic leukemia77.4±17.36HL60Promyelogenous leukemia30.7±3.17K562Erythroleukemia27.9±0.58HEL92.1.7Erythroleukemia11.4±5.89Meg01Megakaryocytic leukemia33.0±3.33

5-Aza-2′-deoxycytidine (decitabine) can relieve p21WAF1 repression in human acute myeloid leukemia by a mechanism involving release of histone deacetylase 1 (HDAC1) without requiring p21WAF1 promoter demethylation

Decitabine is a potent demethylating agent that exhibits clinical activity against myeloid malignancies. Numerous genes silenced by hypermethylation are reactivated by decitabine through a mechanism involving promoter demethylation with subsequent release of histone deacetylases (HDACs) and accumulation of acetylated histones. Recent studies indicating that decitabine also induces regional chromatin remodeling of some unmethylated genes suggest additional mechanisms of action. Decitabine reactivates unmethylated p21WAF1 in some AML cell lines but the possible occurrence of p21WAF1 methylation in AML in vivo has not been studied in detail and decitabine effects on p21WAF1 chromatin remodeling have not been reported. We found that p21WAF1 mRNA was undetectable in 6 of 24 AML patient samples and 4 of 5 AML cell lines but there was no evidence of p21WAF1 promoter methylation. However, decitabine induced p21WAF1 in AML cell lines KG-1 and KG-1a in association with release of HDAC1 and increased acetylated histone H3 at the unmethylated p21WAF1 promoter. Decitabine effects on p21WAF1 histone acetylation and induction were enhanced by the HDAC inhibitor trichostatin A and were independent of wild type p53. Our findings indicate that decitabine can relieve p21WAF1 repression in AML by a mechanism that involves release of HDAC1 without requiring promoter demethylation. Furthermore, our study provides evidence that combined decitabine and HDAC inhibitor treatment can enhance chromatin remodeling and reactivation of an unmethylated tumor suppressor gene. This latter finding is of relevance to the clinical use of these agents in AML as we found the p21WAF1 promoter to be unmethylated in vivo.

Inactivation of the tissue inhibitor of metalloproteinases-2 gene by promoter hypermethylation in lymphoid malignancies

The tissue inhibitor of metalloproteinases-2 (TIMP-2) is known to antagonize matrix metalloproteinase activity and to suppress tumor growth, angiogenesis, invasion and metastasis. We analysed the methylation status of the CpG island in the TIMP-2 promoter region by methylation-specific polymerase chain reaction (MSP) in hematopoietic cell lines. TIMP-2 promoter hypermethylation in the lymphoma cell line Raji and the leukemia cell line KG1a was associated with transcriptional repression. Treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in TIMP-2 upregulation in both cell lines. TIMP-2 was expressed in the cell lines HL60, U266 and XG1, which carry an unmethylated promoter region. MSP analysis of primary patient samples revealed aberrant methylation of TIMP-2 in 33/90 (36.7%) cases of non-Hodgkin's lymphoma (NHL), but not in normal peripheral blood lymphocytes as well as in nonmalignant bone marrow and lymph nodes. The frequency of TIMP-2 methylation was slightly higher in aggressive NHL subtypes compared to those with an indolent subtype (38.6 versus 33.3%). In contrast, TIMP-2 was not hypermethylated in any of the 40 cases of acute myelogenous leukemia examined. We conclude that promoter hypermethylation of TIMP-2 is a novel epigenetic event in the pathogenesis of lymphoid malignancies and may contribute to a more aggressive NHL phenotype.

A combination of catalase and trehalose as additives to conventional freezing medium results in improved cryoprotection of human hematopoietic cells with reference to in vitro migration and adhesion properties

Cryopreservation of hematopoietic cells from cord blood is an essential component in unrelated transplant settings. Cell damage during freezing is caused by multiple factors, of which membrane damage and oxygen free radical generation are two major factors. It was reported earlier that a combination of catalase and trehalose as additives in freezing medium affords better cryoprotection in terms of long-term culture assays. Mononuclear and CD34+ cells isolated from cord blood were used as a source of hematopoietic cells. KG1a cell line was used as a model system in adhesion assays. The cells were frozen in a programmable freezer and stored at -196 degrees C. Various homing-related assays were carried out on the frozen cells. Herein it is reported that these two additives afford better preservation of adhesion- and migration-related properties of the frozen cells. The test cells frozen with additives resulted in improved migration toward stromal-derived factor-1alpha and showed higher expression of its receptor CXCR4. Colony-forming unit assay of migrated test cells showed that these cells are early progenitors having capacity to give rise to all types of myeloid colonies. Test cells also show increased expression of FLT3R and improved responsiveness to FLT3 ligand, the homing-related cytokine. Adhesion to stroma and extracellular matrix was better in test cells as compared to control cells. The present data provide evidence that addition of catalase and trehalose to the conventional freezing medium preserves migration- and adhesion-related properties of the hematopoietic graft.

AML1/ETO Expresssion in Myeloid Leukemia Cells Is Associated with Enhanced Growth-Inhibitory and P15/INK4b Demethylating Effects of 5-AZA-2′-Deoxycytidine.

The chromosomal translocation (8;21) results in the expression of the chimeric transcription factor AML1/ETO, the most frequent fusion gene in acute myeloid leukemia. The AML1/ETO fusion protein acts as a transcriptional repressor by mediating epigenetic silencing through recruitment of histone deacetylases. Recently, it was shown that it also mediates gene silencing by associating with DNA methyltransferase (Dnmt). We therefore hypothesized that cells expressing AML1/ETO might be preferentially sensitive to the effects of an inhibitor of Dnmt activity, and might provide a superior model for in vitro demethylation and reactivation of the promoter of the p15/INK4b gene (encoding a negative regulator of the cell cycle) that is frequently methylated and silenced in AML and MDS.The 3 myeloid cell lines Kasumi-1 cells (AML1/ETO-positive), KG-1, and KG-1a (both AML1-ETO-negative) are all bearing a heavily methylated p15/INK4b promoter. They were treated with 50 – 1000 nM 5-aza-2′-deoxycytidine (DAC) for three pulses of 24 hrs each. After 6 days, cell growth and viability were determined and FACS analysis performed after propidium iodide staining. Kasumi-1 showed the highest sensitivity to DAC treatment (growth inhibition at 500 nM DAC: Kasumi-1 74.28 %, KG-1 69.16 %, KG-1a 62.38 %). In addition, DAC treatment (500 nM) led to a stronger increase in the sub-G1 fraction in Kasumi-1 (30.46%) compared to KG-1a (20.84 %).Regional p15/INK4b promoter methylation was assessed quantitatively by bisulfite sequencing of ≤10 individual cloned alleles (containing 21 CpGs residues) for calculation of methylated CpG percentage. The p15/INK4b was highly methylated in all 3 cell lines (methylated CpGs Kasumi-1 95.2 %; KG-1 89.6 %; KG-1a 98.4 % ). In Kasumi-1 cells, treatment with DAC resulted in a striking, dose-dependent regional demethylation of the p15/INK4b promoter (demethylated CpGs at 200 nM of DAC: Kasumi-1 63.8 %, KG-1 48.9 %, KG-1a 9.3 %). No demethylating effect was achieved with equitoxic doses of cytarabine or melphalan. Effective demethylation of the p15/INK4b promoter was associated with p15/INK4b protein induction as determined by Western Blot. Simultaneous treatment with all-trans retinoic acid (ATRA) enhanced the effects of DAC treatment upon growth inhibition, but not upon p15/INK4b induction. U937 cells with ecdysone inducible AML1/ETO expression (Fliegauf et al, Oncogene 2004) were also treated with different doses of DAC. When AML1/ETO was induced, U937 cells showed a higher growth inhibition (U937 + AML1/ETO 38.6 %, U937 - AML1/ETO 18 % at 25 nM) and increase in Sub-G1 (U937 + AML1/ETO 18 %, U937 - AML1/ETO 10.55 % at 100 nM) after treatment with DAC.Our results imply that the growth-inhibitory and proapoptotic effect of DAC on leukemia cells is modulated by AML1/ETO protein (or its target genes). The greater accessibility of the p15/INK4b promoter to the demethylating effect of DAC in AML1/ETO expressing Kasumi-1 cells may also be due to differences in regional chromatin structure. With their differential sensitivity to DAC, the cell lines Kasumi-1 and KG-1a provide a model for the different responses of leukemic blasts to DAC.

Both 5′ and 3′ Distal Regulatory Elements Are Required for Human CD34 Gene Expression.

In order to understand the controls of gene expression in hematopoietic stem cells (HSC), we have studied the regulation of human CD34, a cell surface glycoprotein used in isolation of HSC. We previously reported that regions 10 to 18 kb 5′ upstream of the transcription start site (TSS) and/or 17 to 26 kb 3′ downstream of the transcription termination site (TTS) of the human CD34 gene contain critical elements for expression in vivo. Mice transgenic for a human CD34 PAC clone (PAC54A19) that includes 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequences expressed human CD34 antigen in the majority of HSC. Because these transgenic mice also demonstrated downregulation of human CD34 gene expression with maturation of hematopoietic cells, we concluded that these critical cis-elements are necessary not only for human CD34 expression in immature cells, but also for downregulation in mature cells. To further delineate the distal cis-elements which regulate the human CD34 gene, we generated two deletion constructs which removed sequences either upstream from −12.8 kb (PmeI deletion) or downstream of +18.2 kb (FseI deletion) of the human CD34 gene. We used a murine myeloblastic cell line, 416B, to test these constructs. We first generated stable clones of 416B cells with PAC54A19; these stable clones expressed human CD34 RNA and protein. In contrast, neither PmeI nor FseI deletion constructs conferred human CD34 mRNA or protein expression in these 416B stable cell lines. Therefore, we conclude that both 5′ upstream and 3′ downstream distal cis-elements are necessary for proper human CD34 expression in cell lines. We also performed DNaseI hypersensitivity assays to localize open chromatin structures in the human CD34 gene in several human myeloblastic cell lines. We detected one DNaseI hypersensitive site (DHS) −12.5 kb upstream of the TSS and several DHS in the region located between +17 kb to +26 kb 3′ downstream of the human CD34 TTS in the human myeloblastic CD34+ KG1a cell line. In addition, we performed similarity searches of the −18 kb to −10 kb human CD34 5′ flanking sequence and +17 kb to +26 kb human CD34 3′ flanking sequence, comparing with the murine CD34 genomic sequence. We identified one conserved region in the −11.5 kb 5′ upstream region of the TSS and one highly conserved region located +19 kb 3′ downstream region of the TTS. To further localize distal cis-elements, we generated several deletion constructs in the parental PAC54A19 PAC clone using the ET cloning system, which facilitates manipulation of PACs by homologous recombination in bacteria. Using this method, we showed that deletion of a 600 bp region located 15 kb 5′ of the TSS or a 1000 bp sequence located 19 kb downstream of the human CD34 gene led to loss of human CD34 expression in 416B stable lines. Our current efforts are focused on identifying the transcription factors that bind to these regions to regulate the expression of human CD34 in stem cells.

A Novel Fusion, KIAA0379 (Ankyrin Repeat Domain 28) to NUP98 in an Adult MDS/AML with a t(3;5;11)(p25;q35;p15).

In hematologic malignancies, deletion of 3p25-26 is a rare but recurrent cytogenetic aberration, indicating the presence of tumor suppressor gene (TSG) on this choromosome arm.We revealed a t(3;5)(p25;q35) that was identified by conventional cytogenetics results in a novel fusion of KIAA0379 on 3p25 to NUP98 on 11p15 in a patient with refractory MDS/AML by the molecular analysis. Fluorescence in situ hybridization (FISH) analysis using whole chromosomal painting and telomeric probes unclosed a criptic three-way translocation t(3;5;11)(p25;q35;p15). FISH restriction using BAC contig revealed that the 3p25 breakpoint is involved in KIAA0379 which locates between BAC 792N12 and 259O19. Split signals of BAC 265K23 for 5q35 and 348A20 for 11p15 showed that NSD1 on 5q35 and NUP98 on 11p15 are the responsible genes of this translocation. As the results of this translocation, expression of three chimeric transcripts were identified by RT-PCR. In KIAA0379-NUP98, exon 18 of KIAA0379 was fused in-flame with exon 13 of NUP98. In NUP98-NSD1, exon 12 of NUP98 was fused in-flame with exon 7 of NSD1. NSD1-KIAA0379 resulted in out-flame fusion. To investigate the expression of KIAA0379 in leukemic cells, RT-PCR for full-length KIAA0379 transcript was performed on bone marrow cells from the patient and a healthy volunteer, and leukemic cell line K562, HL-60 and KG-1a cells. Full-lengh PCR product with expected size was found in all samples, however, short variant transcript was amplified significantly from KG-1a and HL-60 cells. Subsequent sequence analysis of the short PCR product from KG-1a cells revealed a direct fusion of a part of exon 10 to a part of exon 28 without any hidden splicing sites. Southern blot analysis using a cloned KIAA0379 cDNA fragment suggested an internal deletion of KIAA0379 in KG-1a cells. KIAA0379 locates at 3p25.1 about 5.5Mb centromeric to VHL and is predicted to have 28 ankyrin repeat motifs by computational analysis. It is thought to be involved in protein to protein interaction whereas its precise funtion remains unclear. Immunostaining for KIAA0379 protein in NIH3T3 cells transfected with the wild KIAA0379 and the short variant KIAA0379 revealed that the wild KIAA0379 protein localized in cytoplasm and nuclear membrane, on the other hand, the truncated KIAA0379 protein localized exclusively on nuclear membrane. KIAA0379-NUP98 fusion protein is expected to localize on nuclear membrane by sub-localization signal of C-terminal of NUP98. Therefore, dysfunction of KIAA0379 protein might lead to leukemogenesis and KIAA0379 may be potential candidate for a novel TSG. In addition, it has been reported that t(5;11)(q35;p15.5) results in NUP98-NSD1 fusion in childhood AML. To our knowledge, this is the first report in adult case.

Targeting of mTOR in AML Is Associated with Decreased Growth of Leukemic Cells and Downregulation of VEGF.

The mammalian target of rapamycin (mTOR), a key-regulator of cell cycle progression, has recently been implicated in growth of neoplastic cells and tumor- and leukemia-associated angiogenesis. In addition, mTOR has been described as a regulator of expression of vascular endothelial growth factor (VEGF), a cytokine involved in tumor-related angiogenesis, in various neoplasms. We asked whether mTOR can be employed as a new therapeutic target in acute myeloid leukemia (AML) using the mTOR-targeting drug rapamycin and its derivatives RAD001 (everolimus) and CCI-779. As assessed by 3H-thymidine incorporation, rapamycin was found to counteract growth of AML cells in vitro in 9/11 patients examined as well as in the AML-derived cell lines U937, HL60, and KG1a. The effects of rapamycin on growth of AML cells were dose-dependent (primary AML cells: IC50: 10 pM - 1 nM; inhibition of 3H-thymidine uptake at 1 nM of rapamycin: 43±15 % of control; p<0.05) and associated with G1 cell cycle arrest as well as apoptosis. The same results were obtained with RAD001 and CCI-779. In all responsive cells analyzed, rapamycin and its derivatives decreased expression of VEGF in AML cells at the mRNA- and protein level. Exogenously added VEGF was found to revert the rapamycin-induced inhibition of growth in HL60 cells, but did not revert rapamycin effects in KG1a cells, U937 cells, or primary AML cells. In summary, our data suggest that rapamycin and its derivatives act antileukemic in AML cells by two distinct mechanisms, namely direct inhibition of cell growth and suppression of proangiogenic VEGF. mTOR may be a new interesting target in AML.