bluuebunny/crossref_metadata_2025 huggingface dataset:
Study on Telomerase Activity of Human Bone Marrow Mesenchymal Stem Cells.
Authors: Li Jingyuan , Lai Xiaoyu , He Huang
Published: April 2020

Human mesenchymal stem cells(hMSCs) have multiple differentiate potential, and it can differentiate into adipocytes, osteogenic cells, chondrocyte and neural cells et al. It has been reported that telomerase activity in hMSCs is negative, but it is still controversial and telomerase activity in hMSCs-derived adipocytes has not been reported. We investigate the telomerase activity in hMSCs before and after their committed differentiation into adipocytes in vitro and cryopreservation. hMSCs were isolated from normal human bone marrow fellowed by cell culture in DMEM with low glucose containing 10% FBS. The FACS was performed to examine the expression of cell surface molecules and analyze cell cycle of primary hMSCs.Then some of hMSCs were induced to differentiate into adipocytes in vitro by being treated with adipocytic medium fellowed by being stained with oil red O, and the others were cryopreserved in liguid nitrogon for three months. TRAP assay(telomerase repeat amplification protocol assay)was employed to detect telomerase activity in those hMSCs. T293 cells and α-Interferon were analyzed with each test as an additional positive control and negative control respectively. Telomerase activity was expressed as a percentage of the relative telomerase activity (RTA) of the hMSCs relative to the RTA of T293 cells. The results indicated the cells were positive for SH2, SH3, CD90 and negative for CD34, CD45, AC133. It was showed that the majority of primary hMSCs(85%) was at cell cycle of G0/G1 phase and the minority of hMSCs was at S, G2 or M phase. 80% hMSCs was orange adipocytes after they were treated with adipocytic medium for 3–4weeks. Telomerase activity was negative in hMSCs both at the beginning of culture and at the later stages during cell expansion,telomerase activity in hMSCs-passage 1–3(n=10) and hMSCs-passage 4–7(n=9) made no significant difference(1.46±0.83% vs 1.46±0.67%, p=0.99). Cryopreservation did not affect the telomerase activity in hMSCs. Telomerase activity in fresh hMSCs(n=13) and frozen hMSCs(n=6) made no significant difference(1.41±0.44% vs 1.51±1.07%, p=0.64). Telomerase activity in hMSCs-derived adipocytes(n=3) was significantly higher than in hMSCs(n=19)( 11.8±2.52% vs 1.46. ±0.67%, p<0.00001). It is concluded that hMSCs are telomerase-negative, and the stage of culture or cryopreservation does not affect their telomerase activity. After being induced to differentiated into adipocytes, hMSCs telomerase activity is upregulated.
High Efficiency Transduction of Human Mesenchymal Stem Cells Using Retroviral Gene Transfer with Triple Reporter Genes.
Authors: Niu Ting , Najjar Amer M. , Robinson Simon N. , Yang Hong , Decker William K. , McMannis John D. , Ng Jingjing , Thomas Michael W. , Xing Dongxia , Champlin Richard E. , Gelovani Juri G. , Shpall Elizabeth J.
Published: April 2020

Cotransplantation of ex vivo-expanded mesenchymal stem cells (MSC) together with hematopoietic stem cells hastens hematopoietic recovery in animal models and potentially humans. The in vivo mechanisms of migration, homing and long-term viability of MSC following implantation are not well understood. Reporter gene labeling would permit the tracking of transplanted MSC in vivo through noninvasive imaging techniques, namely fluorescence, bioluminescence, and nuclear imaging. Accordingly, a gibbon ape leukemia virus (GALV)-pseudotyped retrovirus was utilized to transfer a triple fusion gene reporter system, expressing enhanced green fluorescent protein (eGFP), luciferase, and herpes simplex virus type one thymidine kinase (HSV1-tk) into MSC. Human MSC were isolated and propagated from normal bone marrow aspirates by culturing ficoll-separated fractions in alpha-MEM medium supplemented with 20% fetal bovine serum. FACS analysis confirmed the MSC phenotype, with cells expressing HLA-Class I, CD105, CD73, CD90 and CD166 antigens and not expressing HLA-Class II, CD80, CD31, CD34 and CD45 antigens. No significant phenotypic changes were observed in early and late passage numbers of MSC. MSC were transduced during their exponential growth phase (as determined by growth curve measurements), with virus-containing medium (VCM) from the PG13 packaging cells (targeting Pit-1 receptors) for 24 hours in the presence of different concentrations of the cationic polymer, polybrene, ranging from 2 μg/ml to 12μg/ml. The 2μg/ml concentration appeared to be optimal for transduction of MSC. VCM was harvested following a 24-hour incubation of the PG13 packaging cells at 32°C or 37°C. These initial experiments suggested that incubation at 32°C appeared to be optimal for virus production. Repeated VCM exposure at 24-hour intervals for 2 or 3 days did not increase the percentage of transduced cells as determined by flow cytometry (eGFP-positive). VCM harvested at 32°C added to MSC in the presence of 2μg/ml polybrene, yielded a 62% transduction efficiency following one round of exposure. U87 human glioma cells were used throughout as a positive control to verify retrovirus efficacy and demonstrated >90% transduction efficiency. MSC clones highly expressing triple reporter genes are being selected and propagated in our laboratory to image transduced cells in NOD/SCID mice, thereby providing a better understanding of the potential role of these cells in transplantation processes.
Superiority of 3-D Hematopoietic Stem Cell (HSC) & Stroma Cell Co-Cultures in Supporting the Proliferation of Transplantable HSC.
Authors: Meretzki Shai , Kadouri Avinoam , Zipori Dov , Berger Ora , Rowe Jacob M.
Published: April 2020

Stem cells are intimately associated in-vivo with discrete niches within the 3-dimensional (3-D) bone marrow micro-environment collectively provide molecular signals that mediate their differentiation and self-renewal. A recently developed Plug-Flow Bioreactor System (PluriXTM), which closely mimics the physiological bone marrow environment by combining 3-D stromal cultures with a continuous flow system, is capable of supporting long term maintenance/expansion of transplantable human HSC.

The stroma cells, growing inside the PluriXTM bioreactor on porrosive carriers enable the propagation of high density (>100-fold input) 3-D stroma cell cultures for prolonged periods of time. Within 40 days, the number of primary human stroma cells seeded in the bioreactor at a density of 5*104 cells/ml reached 1.5*106 cells/ml and the stroma cell lines reached a density of 5*107–1*108 cells/ml. Three-dimensional static stroma HSC co-cultures were found to be superior to 2-D co-cultures in supporting the maintenance of CB HSC and early hematopoietic progenitors.

When cord blood CD34+ cells were seeded on 2-D & 3-D stroma cell static cultures, the 3-D stroma cultures proved superior in supporting the growth of CD34+, CD34+38− and CD34+38−CXCR4+ cells. The 3-D primary human stroma static cultures supported these cells 4.7, 22, 7.4 fold better than the 2-D cultures, respectively.

The CD34+ cells seeded on the high-density 3-D stroma cell cultures within the PluriXTM bioreactor demonstrated significant superiority to 2-D and 3-D stroma cell static cultures and 2-D stroma cultures in supporting the growth and expansion of HSC and earlier hematopoietic progenitors.

These findings verify that a system based on 3-D stroma co-cultures is preferable for ex-vivo expansion of human HSC’s. The PluriXTM system offers wide-range potential for expansion of stem cells from cord blood and production of specific blood products.
Altered Replication Timing of IGH Alleles in Burkitt’s Lymphoma Depends on IGH Breakpoint Position.
Authors: Guikema Jeroen E.J. , de Boer Conny , Gadiot Jules , Schuuring Ed , Kluin Philip M.
Published: April 2020

Monoallelic expression of immunoglobulin genes is epigenetically regulated and maintained. Asynchronous replication of the IGH alleles has been implicated in allelic exclusion. Usually, the functional IGH allele replicates early in the S-phase of the cell cycle whereas the non-functional allele replicates late. Previously, the intronic enhancer region (Eμ) and the 3′ Cα enhancer region have been designated as putative replication initiation sites. Activity of these replication origins are likely to be involved in regulation of asynchronous replication. By use of interphase and DNA fiber FISH we have performed a detailed analysis of the configuration of the t(8;14) chromosomal translocation in Burkitt’s lymphoma patients and cell lines and showed that the breakpoints in all studied cases were perfectly reciprocal without loss of IGH genomic material. An important implication is that in patients and cell lines harboring a breakpoint in a downstream switch region (Sγ or Sα) the Eμ and the 3′ Cα enhancer region are physically separated from each other, whereas in patients and cell lines with the IGH breakpoint located in the JH-region both enhancer regions remain on the der(14) chromosome. We therefore studied the IGH replication timing in Burkitt’s lymphoma cell lines harboring different IGH breakpoints by use of BrdU-FISH and interphase FISH on sorted cell cycle fractions. In two cell lines with JH-region breakpoints (Jiyoye and DG-75) the t(8;14) was invariably targeted to the late replicating IGH allele. In contrast, in all three cell lines with switch-region breakpoints (CA-46, Namalwa, BL-65) asynchronous replication of the IGH alleles was lost. As the position of the breakpoint in the MYC locus at 8q24 differed substantially between these cell lines (DG-75 and CA-46: intron 1 of the c-myc gene; Jiyoye, Namalwa, and BL-65: 100 kb to >500 kb centromeric from c-myc) it is unlikely that the position of the 8q24 breakpoint is of crucial influence on IGH replication timing. We speculate that the Eμ and the 3′ Cα enhancer region regulate the IGH replication timing in a cis-acting manner as physical separation of both enhancer regions results in loss of asynchronous replication.
Evaluation of Focal Adhesion Kinase Gene Expression in Leukemia and Cancer Cell Lines Using Combined Molecular Cytogenetic Investigations and Quantitative Real Time RT-PCR.
Authors: Campos Lydia , Tchirkov Andrei , Cornillon Jerome , Mihaescu Mirella , Giollant Michel , Piselli Simone , Vago Philippe , Cottier Michele
Published: April 2020

FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase protein implicated in integrin-mediated signal pathways. The human gene fak is located on chromosome 8q near c-myc. Cells that overexpress FAK show increased migration and increased cell survival with apoptosis inhibition. The aim of the present study was to investigate the genomic or post-genomic origin of this overexpression in seven tumor cell lines with different levels of FAK expression: K562, an erythroblastic cell line; U937, a monoblastic cell line; KG1a, a poorly differenciated myeloblastic cell line; HL 60, a promyelocytic cell line; Jurkat, a lymphoblastic cell line; A549, a bronchial adenocarcinoma cell line; M4Beu, a lymph node metastasis melanoma cell line. Total or partial chromosome 8 qualitative or quantitative abnormalities and the presence of double minute chromosomes bearing coding genomic DNA fragments were investigated using both conventional cytogenetic (standard karyotype after R- and G- banding) and molecular cytogenetic such as multicolor karyotype (M-FISH), FISH and comparative genomic hybridization (CGH). The mRNA expression was investigated using quantitative real time RT-PCR and the protein level using immunocytochemistry and western blot (immunoblot) analysis. Except for the Jurkat cell line, karyotypes revealed complex modifications. FISH and M-FISH allowed to precisely identify the chromosomal origin of the genomic material on each chromosome and CGH allowed to characterize relative loss and gain of coding genomic DNA and to identify their chromosomal origin. Summarized results are shown in the following table:

No detectable RT-PCR product was observed for HL60 and U937 cell lines. The preliminary results show that there was usually a good correlation between PCR data and western blot analysis. However, FAK overexpression for KG1a cell line most probably has a genomic origin, while in K562 and A549 cell lines the origin is most probably at the transcription or post-transcription level without any abnormality of chromosome 8. Dysregulation of fak gene is observed in hematopoietic as well as solid tulor cell lines, and may contribute to leukemogenesis.

Cell line FISH 8q24-qter CGH Chr. 8 RT-PCR RT-Q-PCR Western blot RT-Q-PCR results in copy number ratio K562 Normal Normal + 130.1 ++++ KG1a Amplified enh(8)(q11qter) + 37.5 ++++ Jurkat Normal Normal + 8.7 + (low) U937 Normal enh(8)(pterqter) 0 0 0 HL60 Amplified amp(8)(q23-24) 0 0 0 M4Beu Normal Normal + 43.5 0 A549 Normal Normal + 46 ++++
High Expression of Micro-RNA BIC / miR155 in All Subtypes of Hodgkin Lymphoma.
Authors: van den Berg Anke , Kluiver Joost , de Jong Debora , Koekoek Theresia , Blokzijl Tjasso , Poppema Sibrand , Kroesen Bart-Jan
Published: April 2020

We recently demonstrated that the noncoding BIC gene was highly expressed in Hodgkin Reed-Sternberg (HRS) cells of the vast majority of both classical and nodular lymphocyte predominant (NLP) Hodgkin lymphoma (HL). The BIC gene belongs to the family of noncoding mRNA-like molecules and was suggested to function through its noncoding RNA transcript. The human BIC gene shows a high degree of sequence identity to the Mouse and Gallus gallus BIC genes, which was most pronounced in a region containing a ~70 nucleotide stem-loop structure. Recently published data revealed expression of miR155 micro-RNA in murine tissue. The miR155 sequence was identical to the first 22 nucleotides of the ~70 nucleotide stem-loop structure of the BIC gene indicating that BIC is a primary micro-RNA transcript. Micro-RNAs (miRNA) are endogenous small RNAs (20–22 nucleotides) that inhibit protein translation by paring to the messages of protein-coding mRNAs. Based on recent data, it is clear that deregulation of miRNA expression, processing or stability may lead to deregulation of important cellular processes and thereby contribute to the development of cancer.

In this study we analyzed the expression of miR155 in HL and NHL samples and studied the regulation of BIC/miR155 expression. Northern blot analysis revealed that HL cell lines, which all have a high level of BIC transcripts, also have high levels of the functionally mature miR155. NHL samples with low levels of BIC transcripts also have low levels of miR155. These data provide experimental support for processing of primary BIC transcripts to the functionally mature miR155 fragments and indicate that miR155 expression is specific for HL. To analyze whether BIC expression could be induced in NHL cell lines we studied several stimuli in the wild type Burkitt lymphoma derived cell line Ramos and a NF-κB deficient variant cell line. Real-time RT-PCR analysis revealed a high level of BIC and of the 70 nucleotide miR155 precursor in anti-IgM stimulated Ramos cells. Anti-IgM stimulation of the NF-κB deficient Ramos cell line revealed only low levels of BIC and pre-miR155. These data demonstrate that transactivation of NF-κB is required for continuous upregulation of BIC in Ramos cells. Stimulation of Ramos cells with PMA/calcium-ionophore, which directly activates PKC, significantly upregulated the level of BIC and pre-miR155. This suggests that activation of PKC and its downstream signaling components, including NF-κB are involved in the regulation of BIC expression. Thus, whereas HRS cells typically do not express surface immunoglobulin, upregulation of BIC in these cells may be induced by constitutively activated NF-κB.
Microarray Analysis of the Short Isoform of Ikaros Overexpressed in Patients with Hematologic Malignancies.
Authors: Kataoka Itaru , Ishimaru Fumihiko , Tanimoto Mitsune
Published: April 2020

We analyzed expression of the transcription factor Ikaros in human hematologic malignancies and found overexpression of the short isoform of Ikaros, Ik-6, in patients with blast crisis of chronic myeloid leukemia and pre-B cell acute lymphoblastic leukemia. We have made several stable transfectants which overexpress Ik-6 and analyzed the consequence of Ik-6 overexpression.

First, overexpression of Ik-6 in FDH-1, which was established from a patient with early pre-B acute lymphoblastic leukemia, and Ramos conferred resistance to dexamethasone- and anti-IgM-induced apoptosis, respectively. Upon treatment with dexamethasone, FDH-1 cells demonstrated massive apoptosis measured by staining with Annexin V. In contrast to mock transfectants, Ik-6 transfectants showed less marked apoptosis (mock 86.4% vs Ik-6 34.4–73.6%, p=0.01). We also used Ramos as a model of anti-IgM-induced apoptosis, and flow cytometric analysis was performed with Annexin-V staining. In contrast to mock transfectants, Ik-6 transfectants showed less pronounced apoptosis (mock 40.4% vs Ik-6 23.1–27.4%, p=0.03). Next, overexpression of Ik-6 in Nalm6 lead to impaired cell growth and cell cycle progression. Cell growth and cell cycle were analyzed and compared with mock transfectants and Ik-6 transfectants using MTT assay and DNA content analysis with PI staining, respectively. MTT assay demonstrated that, after three days of growth, Ik-6 transfectants grew more slowly than mock transfectants (Ik-6/mock ratio 0.62–0.67; p=0.01). DNA content analysis showed that, after three days of growth, more Ik-6 transfectants underwent G0–G1 arrest than mock transfectants (Ik-6/mock ratio 1.15–1.21; p=0.01), and less Ik-6 transfectants entered into S phase than mock transfectants (Ik-6/mock ratio 0.84–0.90; p=0.05).

Finally, overexpression of Ik-6 promoted all-trans retinoic acid (ATRA)-induced granulocytic differentiation, and, on the other hand, inhibited vitamin D3 (VD3)-induced monocytic differentiation of HL-60. Upon induction of granulocytic differentiation by ATRA, mock transfectants showed 74.5% positivity of myeloperoxidase (MPO) staining. In contrast, Ik-6 transfectants showed 51.5–56.8% of positivity of MPO staining (p=0.04). NBT reduction assay demonstrated 43.8% positivity with mock transfectants and 62.3–67.3% positivity with Ik-6 transfectants (p=0.001). Upon induction of monocytic differentiation by VD3, mock transfectants showed 51.0% positivity of nonspecific esterase (NSE) staining. In contrast, Ik-6 transfectants showed 26.8–36.0% of positivity of NSE staining (p=0.03).

To understand the underlying mechanisms, we report the results of microarray analysis of Ik-6 transfectants.
Distinct Biological Impact of Dephosphorylation vs Downregulation of p210Bcr-Abl: Implications for Imatinib Mesylate Response and Resistance.
Authors: Kurzrock Razelle , Talpaz Moshe , Li Lan , Estrov Zeev
Published: April 2020

Imatinib mesylate acts by suppressing phosphorylation of its kinase target - Bcr-Abl. We hypothesized that loss of p210Bcr-Abl (the kinase target) may lead to imatinib mesylate resistance. Two model systems were studied: K562 cells (CML blast crisis line) and MO7E/MBA-1 cells (with MBA-1 cells representing MO7E cells stably transfected with BCR-ABL). Relative resistance to imatinib mesylate developed when p210Bcr-Abl expression was abolished. Furthermore, K562 cells were significantly more growth suppressed after exposure to imatinib mesylate (p210Bcr-Abl is present but dephosphorylated), than after downregulation of Bcr-Abl expression. Signaling pathways which were functional in the absence of Bcr-Abl expression - NF-KB and MAP kinase activation or the growth factor pathway -- were disrupted when p210Bcr-Abl was present but dephosphorylated, suggesting that an intact, but enzymatically inactive Bcr-Abl, may interfere with critical growth/signaling pathways. Downregulation of p210Bcr-Abl may be a mechanism by which imatinib mesylate-resistance emerges. Samples from 3 of 15 patients with imatinib mesylate-resistant CML blast crisis had undetectable levels of p210Bcr-Abl. We conclude that retention of a dephosphorylated p210Bcr-Abl has a biologic impact distinct from that of downregulation/loss of p210Bcr-Abl and, in a subset of patients, loss of the target of the kinase inhibitor may lead to imatinib mesylate resistance.
Arsenic Trioxide Induced p53-Dependant Apoptotic Signals in a Temperature Sensitive (Ts) p53 Mutant, BRK Cell Line.
Authors: Akay Cagla , Gazitt Yair
Published: April 2020

Mutations in the p53 gene are the most common genetic abnormality in human cancers and are the cause for drug resistance. Arsenic trioxide (ATO) is an effective chemotherapeutic agent for the treatment of acute promyelocytic leukemia (APL) and is being tested in phase II studies in various types of cancers. We have previously shown that ATO is a potent inducer of apoptosis in multiple myeloma cells, engaging the intrinsic apoptotic pathway in cells expressing w.t. p53 whereas in cells expressing mutant p53, both the intrinsic and extrinsic apoptotic pathways are engaged. To further establish the differential effect of ATO in relation to the p53 status we studied the effect of temperature shift in temperature sensitive (Ts)-p53 expressing baby rat kidney (BRK) cells. We studied by Western immunoblotting the activation of the intrinsic and the extrinsic apoptotic pathways in ATO-induced apoptosis of BRK cells cultured at 32°C (w.t. p53 phenotype) and 37°C (mutant p53 phenotype). As expected, at 32°C, we observed a G1 arrest through activation of p21. We also observed depolarization of mitochondrial membrane; the release of cytochrome C and activation of caspase-9 and apoptosis as measured by Annexin V. We also observed release of SMAC at 32°C. In contrast, at 37°C we observed a G2/M arrest with no activation of p21 with activation of the extrinsic apoptotic pathway through early induction of TRAIL and TRAIL receptor- R2, activation of caspase-8, activation of BID, degradation of FLIP, rapid depolarization of mitochondrial membrane and release of AIF from mitochondria to the cytosol. We also demonstrate by flow cytometry and confocal imaging translocation of AIF to the nucleus in ATO-induced apoptosis at 37°C but not at 32°C. These results further substantiate our p53 model (Akay and Gazitt, Cell Cycle2:258–268, 2003).
The Fluctuation of Telomere Repeat Binding Factor 1 during Cell Cycle and Its Localization in Telomerase-Positive and Negative Cells.
Authors: Lan Jianping , Huang He , Zhu Yuanyuan , Sun Jie
Published: April 2020

Telomere is a nucleoprotein complex which caps the extreme ends of eukaryotic chromosomes. In human, telomere is composed of a tandem repeat array of TTAGGG hexanucleotide and bound to a set of specific proteins. These proteins function to maintain the integrity of chromosomes and genomic stability. Among these proteins, telomere repeat binding factor 1(TRF1) is the first telomere binding protein which was isolated by DNA affinity chromatography in 1995. TRF1 serves as a negative regulator of telomere length since TRF1 overexpression would elicit the shortening of telomere length in telomerase-positive cells. Meanwhile, overexpression of TRF1 would also induce the entry into mitosis and increase mitotic cells. These observation indicated TRF1 might participate in cell cycle regulation. However, the underlying mechanism in which TRF1 regulates the cell cycle and the endogenous level of TRF1 were not well-documented during cell cycle progression. To address these questions, we arrested HeLa cells at different phases by a combination of thymidine(5mM at final concentration) and nocodazole(20mM at final concentration) and detected the TRF1 levels by semi-quantitive Western Blotting assay. Cell cycle was verified by flow cytometry. Our results showed TRF1 level fluctuated coincided with cell cycle progression which reached the zenith at the M phase and went down to the nadir at G1/S point. Densitometry analysis demonstrated that the level of TRF1 at M phase was 3.9 times more than that at G1/S point(n=3, p<0.01). These results suggested that TRF1 might be essential for proper cell cycle progression and it was likely to take part in regulation of cell cycle chechpoint. TRF1 is also expressed in telomerase-negative cells. To further discriminate the different functions of TRF1 and decipher its protein-protein interaction network in telomerase-positive and negative cells, full-length TRF1 cDNA was amplified by PCR and subsequently subcloned into pEGFP-C2 vector to express TRF1 tagged by enhanced green fluorescent protein. This construct was then transiently transfected into telomerase-negative cells(WI38-2RA) and telomerase-positive cells(HeLa). Immunoflourescent staining was employed to check the localization of TRF1 in these two kinds of cells. Although in both cells, TRF1 was distributed in a speckled pattern in the nuclei, TRF1 did exclusively colocalize with promyelocytic leukemia(PML) nuclear body in WI38-2RA cells but not in HeLa cells. PML fused with RARα due to chromosome15,17 translocation which led to disassembly of PML nucleur body in acute promyelocytic leukemia. These preliminary results suggested that TRF1 might have the different regulating mechanism and interacting network.
Overexpression of Par-4 Increases TRAIL-Induced Apoptosis in Neoplastic Lymphocytes by Diminishing Expression of Inhibitors-of-Apoptosis-Proteins and by Enforcing Activation of Initiator- and Executionercaspases.
Authors: Boehrer Simone , Nowak Daniel , Hoelzer Dieter , Mitrou Paris S. , Chow Kai Uwe
Published: April 2020

Acknowledging the previously demonstrated deregulated expression of the prostate-apoptosis-response-gene-4 (par-4) in ex vivo cells of patients suffering from acute and chronic lymphatic leukemias, we hypothesized that expression of par-4 determines sensitivity of neoplastic lymphocytes towards TRAIL-induced apoptosis. Evaluating this hypothesis we thus demonstrate, that par-4-transfected Jurkat cells incubated with the agonistic TRAIL-antibody CH11 exhibit a considerably increased rate of apoptosis as compared to their par-4-negative counterparts. Outlining the underlying molecular mechanisms we provide evidence, that par-4 promotes activation of the initiator caspase-8. Despite upregulating Bcl-2, overexpression of par-4 enhances cleavage of Bid and activation of caspase-9, resulting in an enforced activation of the executioner caspases-6 and -7, whereas caspase-3 remains unaltered. These effects are observed with a concomittant down-regulation of the inhibitor-of-apoptosis proteins cIAP-1 and XIAP. In conclusion, we here provide first evidence that expression of par-4 augments sensitivity of neoplastic lymphocytes to TRAIL-induced cell death, and describe the impact of par-4-expression on key molecules considered crucial in the induction of apoptosis via the extrinsic pathway.
Identification of a New t(4;11) Rearrangement Involving MLL and ArgBP2 in a Case of Infant Acute Myeloid Leukemia.
Authors: Tonelli Roberto , Nigro Luca Lo , Serravalle Salvatore , Montemurro Luca , Izzi Giancarlo , Pession Andrea
Published: April 2020

Rearrangement 11q23, involving MLL gene is mainly associated with M4/M5 acute myeloid leukemia (AML) and is characterized by poor prognosis and responsiveness to therapy. In this case blasts from an infant patient with monocytic AML were analyzed at diagnosis. The most frequent translocations of MLL, t(4;11) (40%, MLL-AF4) and t(9;11) (27%, MLL-AF9) were investigated by RT-PCR and resulted negative, meanwhile interphase FISH analysis, performed by using specific MLL probes, revealed a MLL gene translocation. Panhandle-PCR technique was performed to identify the partner gene and candidate clones DNA were sequenced to identify the unknown partner gene. The sequence analysis revealed the presence of a region of MLL connected with a region of ArgBP2 gene (chromosome band 4q35.1). The fusion gene is composed of the first 9 MLL exons and the last 3 ArgBP2 exons (exons 19–21) (Fig. 1). ArgBP2 belongs to vinexines family of protein adapters with three C-terminal SH3 domains, several potential phosphorilation sites for c-Abl and a serine/threonine rich region. The predicted fusion protein consists of AT-hooks and MT domains of MLL and the last ArgBP2 SH3 domain. MLL-ArgBP2 chimeric transcript was expressed in the blast while the reciprocal transcript ArgBP2-MLL was not. Identification of this new translocation of MLL gene confirms the high grade of MLL recombination in infant AMLs. Ongoing studies are in progress to analyze the mechanism leading to this new chromosomal translocation and the leukemogenic potentialities of the new MLL-ArgBP2 fusion protein.

Figure Figure
Specific Inhibition of hTERT by shRNA Sensitizes for Imatinib Mesylate in BCR-ABL-Positive Cells.
Authors: Sashida Goro , Tauchi Tetsuzo , Sumi Masahiko , Nakajima Akihiro , Ohyashiki Junko H. , Ohyashiki Kazuma
Published: April 2020

In the normal somatic cells, loss of telomere length leads to cell senescence appearing chromosome end-to-end fusion, and rearrangements. Most cancer cells exhibit constitutive telomerase activation and maintain telomere stability. Therefore, telomerase produces telomere repeats and represents the anti-cancer therapeutic target. Imatinib mesylate is a tyrosine kinase inhibitor, which inhibits BCR/ABL signal transductions. Despite of high remission rate of imatinib, the resistance to imatinib has been recognized as the major problem in treatment of the advanced or refractory CML. We have demonstrated that a dominant negative form of hTERT presents telomere shortening followed by proliferation arrest and apoptosis in leukemia cell lines. In the present study, we investigated the biological activity of hTERT specific short hairpin RNA (hTERT-shRNA) (kindly gifted from Dr. William C. Hahn, Dana-Farber Cancer Institute) in CML blastic-crisis cell line. We first disrupted the function of a catalytic subunit of telomerase complex, hTERT by expressing the hTERT-shRNA in the telomerase expressing K562 cell line, and found the inhibition of telomerase activity after single cell selections. We also found cumulative telomere shortenings in the hTERT-shRNA-expressing K562 cells. The hTERT-shRNA-expressing K562 cells showed increasing rate of apoptosis, and morphologically hyper-diploid cells followed by proliferation arrest. The hTERT-shRNA-expressing K562 cells reduced number of colony formation. The hTERT-shRNA-expressing K562 cells (population doubling 30) cytogenetically demonstrated end-to-end fusions and multi-centric chromosomes, which might be equivalent to disruptions of telomeric structure composed by repetitive DNA and telomere binding proteins. These results demonstrated that hTERT-shRNA effectively reduced the telomerase activity and induced telomere dysfunction in leukemia cells. Further we evaluated the combination efficacy of hTERT-shRNA and imatinib. The hTERT-shRNA-expressing K562 cells demonstrated significantly higher rate of apoptotic cells, and reduced proliferation activity compared with the parental K562 cells in the dose dependent manner of imatinib. These results suggest that the telomerase inhibition by hTERT-shRNA combined with imatinib is effective in BCR-ABL-positive leukemia.
BCL-6 Overexpression Predicts Poor Response to Reduced Immune Suppression Therapy in Children with Post-Transplantation Lymphoproliferative Disease (PTLD).
Authors: Porter Christopher C. , Liang Xiayuan , Albano Edythe A.
Published: April 2020

Background: PTLD is a well known complication of organ transplantation. While the understanding of this disease is increasing, the diagnosis and treatment remain challenging. The clinical, morphological, and molecular findings in PTLD vary widely. Reduction of immune suppression is the treatment of choice, but is effective in only 20–50% of patients. Mutations in the BCL6 gene have been demonstrated in some lymphomas and PTLD and may have prognostic implications. A single study, in which most subjects were adults, has suggested that BCL6 mutation in PTLD predicts a lack of response to reduced immunosuppression (IS).

Objectives: To determine if BCL-6 overexpression predicts poor response to reduced IS or decreased survival in children with PTLD.

Methods: Clinical data of patients identified with PTLD at The Children’s Hospital (TCH) from 1992–2002 were obtained, including age, type of organ transplanted, subtype of PTLD, treatment received, response to treatment, relapse free survival (RFS), and overall survival (OS). Archived specimens of lesions obtained at the time of diagnosis of PTLD were evaluated for the presence of BCL-6 expression by immunohistochemistry.

Results: Between 1992 and 2002, 486 organ transplants (211 heart, 107 hematopoietic stem cell (HSC), 86 liver, 82 kidney) were performed at TCH. During that same period, 20 children were diagnosed with 21 cases of PTLD. Eleven patients had liver transplants (12.7% of transplants), 5 had heart transplants (2.4%), 3 had HSC transplants (2.8%), and 1 had a kidney transplant (1.2%). Histologic classifications of PTLD included 1 follicular hyperplasia, 9 polymorphic PTLD, and 11 lymphomas. Only 5 of 20 patients responded to reduced IS. Overall mortality was 45% (9/20). BCL-6 was overexpressed in 7 of 18 evaluable specimens from 20 patients. None of the patients with BCL-6 overexpression responded to reduced IS (p=0.05). BCL-6 overexpression was associated with monomorphic histology (p=0.02), but did not predict RFS or OS.

Conclusions: BCL-6 expression is associated with monomorphic histology and non-response to reduced IS in children with PTLD. Larger, prospective studies of BCL-6 mutation and expression are needed to verify the clinical significance of these findings.
Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis through Histone Acetylation and Upregulation of Annexin A1 in AML1-ETO Expressing Kasumi-1 Cells.
Authors: Tabe Yoko , Contractor Rooha , Radke Susanne , Andreeff Michael , Konopleva Marina
Published: April 2020

The t(8;21) translocation, found in 12% of AML, creates the chimeric fusion protein, AML-ETO, which recruits the HDAC complex to AML-1-dependent promoters resulting in transcription repression. Here, we studied the effects of HDAC inhibitor FK228 in the AML1-ETO positive AML cell line, Kasumi-1 and investigated molecular mechanisms of apoptosis induction in these cells. FK228 (5nM, 24 hours) caused marked growth inhibition, apoptosis induction (annexinV+/PI-; control 4.5±0.5%, FK228 24.6±2.6%), and cell cycle arrest (G1 phase; control 59.5%, FK228 76.7%, S phase; control 32.1%, FK228 17.8%). To investigate the molecular changes induced by FK228 we used cDNA array technology. After 24 hour exposure of Kasumi-1 cells, FK228 down-regulated (≤2-fold) 14 genes and up-regulated (≥2-fold) 123 genes including apoptosis and cell cycle regulating genes (Annexin A1; 3.5 fold, serglycin; 2.6 fold, Hes1; 2.2 fold, and TNFα; 2.1 fold increase). Among these genes, we focused on the pro-apoptotic protein Annexin A1, one of the calcium-dependent phospholipid binding proteins. First, we confirmed induction of Annexin A1 mRNA by FK228 using quantitative TaqMan RT-PCR (7.2±1.7 fold). As demonstrated by Western blot analysis, FK228 upregulated the expression of both, full-length Annexin A1 protein and a cleaved isoform of Annexin A1. To identify the critical histone residue modified by FK228 on the Annexin A1 promoter, we performed chromatin immunoprecipitation (ChIP) assays quantified by TaqMan PCR. FK228 increased H4 acetylation (6.7 ±1.8 fold) and H3-K9 acetylation (3.8 ±0.4 fold compared with control at 24 hours) in the Annexin A1 promoter. Next, we investigated effects of FK228 on apoptosis and signal transduction pathways that may modulate Annexin A1 function. As cleavage of the ubiquitous protein Annexin A1 is reported to be associated with caspase activation, the effect of caspase3 inhibitor Z-DEVD-cfm was examined. Z-DEVD pretreatment of Kasumi-1 cells resulted in increased viability and partial inhibition of apoptosis by FK228 (45% decrease in apoptosis with 20μM Z-DEVD). Z-DEVD partially inhibited FK228 induced Annexin A1 cleavage isoform indicating that cleavage of Annexin A1 involves both, caspase-dependent and independent mechanisms. Since activation of the MAPK/ERK pathway has recently been linked to histone de-acetylation, we examined the effects of FK228 on ERK phosphorylation. FK228 used at 5nM inhibited ERK signaling. Further experiments are ongoing to elucidate the possible association of MAPK inhibition, histone acetylation and Annexin A1 gene expression in Kasumi-1 cells.

In summary, these findings suggest that FK228 induces apoptosis and growth inhibition in Kasumi-1 cells in part by inducing Annexin A1 expression through histone acetylation of the promoter in association with caspase-dependent Annexin 1 cleavage and inhibition of MAPK signaling.
Clinical Significance of Prognostic Factors Related to Multidrug Resistance and Apoptosis for Prediction of Chemotherapy Outcomes in Acute Leukemia.
Authors: Ye Xu , Tan Huo , Feng Ying , Su Hang , Pang Ying
Published: April 2020

OBJECTIVE: To study the prognostic value of cd34, P-gp and Bcl-2 expression and MDR functional assays together with patients’ characteristics in patients with de novo acute leukemia.

METHODS: The expression levels of P-gp, CD34, Bcl-2 and the mean fluorescence intensity ratio of rhodamine 123 efflux assay (MIR (RE)) and intracellular daunorubicin accumulation assay (MIR (IDA)) of patients’ leukemia blast samples were prospectively determined with flow cytometry. The results of remission (CR) and non-remission (NR) patients were compared. Patients were given standard chemotherapy for remission induction.

RESULTS: CR patients were comparable to NR patients in age (39.2±23.0: 44.4±22.8, p=0.568, n=30). But NR patients had significantly higher peripheral white blood cell count than CR patients (72.84±78.74: 21.38±17.08, p=0.022, n=30). On the other hand, leukemia blasts samples of CR patients tend to express lower levels of CD34 (22.35±29.20: 46.18±39.5, p=0.084, n=30), P-gp (2.23±2.06: 11.95±22.90, p=0.111, n=30) and Bcl-2 (46.90±35.11: 57.02±31.97, p=0.434, n=30), but not significantly. However, the MIR (RE) (1.16±0.38 vs. 1.43±0.26, P=0.045, n=24) and MIR (IDA) (1.02±0.05 vs. 1.47±0.44, P =0.005, n=24) were both significantly lower in CR patients than in NR patients.

CONCLUSION: Leukemia patients with much higher peripheral WBC count at diagnosis were more difficult to reach remission after induction chemotherapy than patients with lower WBC counts. Age was not influential to chemotherapy results. The determination of MIR value in MDR functional assays is more valuable than expression levels of CD34, P-gp and Bcl-2 in the prediction of chemotherapy outcomes.
Relationship between DNA Index and Cellular Drug Resistance in Children with Newly Diagnosed Acute Lymphoblastic Leukemia.
Authors: Karakas Zeynep , Gokce Selim , Kuruca Serap Erdem , Demirel Gulderen Yanikkaya , Ozbek Ugur , Akcay Arzu , Aydogan Gonul , Devecioglu Omer
Published: April 2020

Leukemic cell ploidy is known risk factor in childhood leukemia. Hypodiploidy (DNA index < 1.00 or chromosome number <46) was shown as adverse prognostic factor in many study. The cause of poor prognosis is not known. Cellular drug resistance might provide important information on this subject. In this study, we want to evaluate LC50 (lethal concentration to 50% of cells) values of 7 different drugs in 34 newly diagnosed patient and compare those of hypodiploid with nonhypodiploid patients. DNA index was determined by flowcytometric analysis of cellular DNA content and cell cycle distribution with Coulter EPICS XL-MCL system on propidium iodide stained cells. DNA index was defined as the modal DNA content of leukemic cells compared with that of reference normal lymphocytes. Patients were divided in hypodiploid (DNA index < 1.00) and nonhypodiploid (DNA index ≥ 1.00)

We carried out in vitro tests with a 4-day culture and a methyl-thiazol- tetrazolium (MTT) assay on bone morrow samples of patients to get LC50 values for 7 different classes of drugs(PRD, prednisolon; DEXA, dexamethasone; VCR, vincristine; ASP, l-asparaginase; ADR, adriamisin; DNR, daunorubisin; ARA-C, cytosine arabinoside) commonly used in treatment of ALL. Eigth patients (23.5%) were belong to hypodiploid group whereas 26 patiens (76.5%) were included in nonhypodiploid group. Sex and age distribution were not different between groups. MTT test were succesfully performed in 7 out 8 hypodiploid patient for each drug. Median LC50 values for PRD and ARA-C were similar. Median LC50 values of hypodiploid group for DEXA, VCR, ASP and ADR were 5.4, 3.5, 5.9 and 3.4 fold higher than those of nonhypodiploid group and were found to be statistically significant. Only the median LC50 value of hypodiploid group for DNR were lower than that of the nonhypodiploid group (p <0.046).

In conclusion, cellular drug resistance might provide important information in relation to poor prognosis of hypodiploid ALL patients. It might also help to individualize the treatment according to drug resistance profiles. Further studies, large study groups and long-term follow-up are needed to confirm these results.
The Expression of mRNA and Enzyme Activity of Deoxycytidine- and Deoxyguanosine Kinases in Cell from Patients with Untreated B-Cell Chronic Lymphocytic Leukemia.
Authors: Lotfi Kourosh , Karlsson Karin , Juliusson Gunnar , Petersona Curt , Eriksson Staffan , Albertioni Freidoun
Published: April 2020

Chronic lymphocytic leukemia (CLL) is the most common leukemia in Europe and North America. The purine analogues fludarabine and cladribine have earned an important place in the treatment of CLL. Yet, the alkylating agent chlorambucil still remains as cornerstone of treatment. A gradual shift towards the use of purine analogues as the central component has become apparent however it is of greatest importance to strictly compare the efficacy of fludarabine and cladribine to have a basis for future clinical studies with these agents. In this study 59 patients included in the international Phase III Trial for untreated B-cell CLL, were randomized in order to evaluate the efficacy of chlorambucil, fludarabine, and cladribine, as primary treatment of patients with symptomatic B-cell CLL. Both fludarabine and cladribine are prodrugs and must be phosphorylated intracellularly to monophosphates by the nuclear/cytosol enzyme deoxycytidine kinase (dCK) and possibly by the mitochondrial enzyme deoxyguanosine kinase (dGK). DCK plays a pivotal roll in the activation of fludarabine and cladribine and it has previously been reported that resistance to these drugs is mainly due to deficiency of the dCK.

Before treatment, peripheral blood cells were isolated and the activity of dCK and dGK enzymes were analyzed in patient cells together with the dCK and dGK mRNA levels using the real-time quantitative PCR method.

The dGK activity was considerably lower (6 fold) than the dCK activity. The enzyme activities in samples from CLL patients varied about 20 and 30 folds for dCK and dGK, respectively.

The mRNA expression for dCK and dGK in patients showed also a large inter individual variability from 0,011 to 0,189 and 0,001 to 0,154 respectively.

There was no correlation between enzyme activity and mRNA levels in the studied CLL patients. We could not found any spliced mRNA dCK variant in our materiel or any mutation, which could explain the discrepancy between dCK activity, and mRNA levels. In conclusion, the results suggest that dCK and dGK expression is regulated at the (post) translational level in leukemic cells. However, studies of the relationship between enzyme activities and/or mRNA levels with clinical response to chemotherapy are underway and will be reported.
Acute Myeloid Leukemia Cells Are Resistant to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (Apo2L/TRAIL) by a Mechanism Independent of Multidrug Resistance.
Authors: Fricke Stacy M. , Minderman Hans , O’Loughlin Kieran L. , Cheng Jinrong , Repasky Elizabeth A. , Baer Maria R.
Published: April 2020

Multidrug resistance (MDR) associated with overexpression of the ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP) is an obstacle to the successful treatment of AML. Apo2L/TRAIL binds to the extracellular death receptors DR4 and DR5 and induces tumor-specific apoptosis in diverse malignancies. Given that Apo2L/TRAIL induces apoptosis via signal transduction pathways, it has the potential to be effective in cells with MDR mediated by ABC proteins. Cell lines with MDR mediated by ABC proteins and acute myeloid leukemia (AML) cells from 10 patients (6 untreated, 4 relapsed/refractory) were tested for expression of the Apo2L/TRAIL receptors DR4 and DR5 and decoy receptors DcR1 and DcR2 by flow cytometry and for cytotoxic and pro-apoptotic effects of Apo2L/TRAIL (Genentech, Inc.). Parental HL60 and 8226 cells and resistant HL60/VCR (Pgp) and HL60/Adr (MRP-1) cells demonstrated expression of DR4 and DR5 and lack of expression of DcR1 and DcR2, while 8226/MR20 (BCRP) had only low-level expression of DR5. Parental HL60 and 8226 cells displayed dose-dependent cytotoxicity in response to Apo2L/TRAIL. HL60/VCR and HL60/Adr were 3- and 5-fold more resistant to Apo2L/TRAIL than parental HL60 cells, but were not sensitized by the Pgp- and MRP-1-specific modulators PSC-833 and MK571, demonstrating that resistance was not mediated by Pgp and MRP-1. 8226/MR20 (BCRP) cells were not resistant to Apo2L/TRAIL in relation to parental 8226 cells. Of note, cyclosporine A (CsA) sensitized all parental and multidrug resistant cells to Apo2L/TRAIL, lowering IC50’s 2- to 5-fold, likely by a pro-apoptotic mechanism independent of MDR modulation. AML blasts did not express Apo2L/TRAIL receptors, with the exception of DR5 in two samples, both DR5 and DcR1 in one and DcR1 alone in a fourth. Moreover, AML blasts showed minimal or no apoptosis following 24- and 48-hour in vitro exposure to Apo2L/TRAIL in concentrations up to 1 ug/ml, 30-fold higher than the highest cytotoxic concentration in cell lines, and no enhancement of apoptosis by CsA. In conclusion, MDR mediated by ABC proteins does not appear to be associated with Apo2L/TRAIL resistance in cell line models, but AML cells are resistant to Apo2L/TRAIL, and this resistance is associated at least in part with lack of receptor expression. Strategies for upregulating Apo2L/TRAIL receptor expression on AML cells warrant further study.