bluuebunny/crossref_metadata_2025 huggingface dataset:
Cells Lacking the PolQ Polymerase Are Moderately Sensitive to Ionizing Radiation and the Oxidant Induced Toxicity of Paraquat and Bleomycin.
Authors: Goff Julie P. , Epperly Michael W. , Shields Donna S. , Smith Tracy , Seki Mineaki , Wang Hong , Wittschieben John P. , Wood Richard D. , Dertinger Stephen D. , Torous Dorothea K. , Greenberger Joel S.
Published: October 2019

The DNA polymerase POLQ (pol theta) can bypass abasic sites in DNA and thymine glycol, a common product of reactive oxygen species damage to DNA. POLQ may also have a back-up role in base excision repair. To explore the role of POLQ in tolerance of DNA damage following irradiation, and exposure to agents that induce oxidative stress we established bone marrow stromal cell lines from the PolQ+/+ and PolQ−/− mice. Irradiation survival curves were obtained for PolQ +/+, and 2 clones of PolQ −/− cells. Dose-response experiments using the free radical generators paraquat, hydrogen peroxide and bleomycin were done on 2 clones each of PolQ +/+ and PolQ −/− cells. There was no significant difference over 23 weeks in cumulative granulopoiesis in LTBMCs from PolQ +/+ and PolQ −/− mice. PolQ+/+ cells and 2 independent clones of PolQ−/− cells were exposed to 0 – 8 Gy of gamma radiation, and colony forming ability was measured. PolQ −/− cells were more sensitive to irradiation as shown by decreased Do from 1.38 + 0.06 Gy for PolQ +/+ cells compared to 1.27 + 0.16 and 0.98 + 0.10 (p = 0.0316) Gy for PolQ −/− clones 1 and 3 respectively. Micronucleated reticulocytes were measured in peripheral blood of PolQ+/+, +/− or −/− mice after irradiation with 75 cGy or 700 cGy. Before irradiation, PolQ−/− mice had a higher fraction of MN- reticulocytes compared to PolQ+/+. Forty hr after irradiation, MN reticulocytes in PolQ−/− mice increased to 8.53 ± 1.48%, compared to 2.35 ± 0.12% and 2.55 ± 0.17% in PolQ+/+ mice (p = 0.0032). PolQ +/+ and PolQ −/− cells were exposed to paraquat (0–20 mM), hydrogen peroxide (0–40 uM) and bleomycin (0–5 ug/ml) for 1 hr prior to plating. After 7 days, the cells were stained and colonies of 50 cells or greater were counted. Statistical analysis was done using ANCOVA. PolQ −/− cells were modestly more sensitive to paraquat (p< 0.0001) and bleomycin (p< 0.0001) than PolQ +/+ cells. When the cells were treated with hydrogen peroxide, there was no significant difference in surviving fraction (p=0.7327) between the PolQ +/+ and the −/− cells. The moderate but significant sensitivity of POLQ-defective cells to bleomycin, ionizing radiation and paraquat supports the hypothesis that POLQ normally participates in helping cells tolerate DNA damage. The increased frequencies of spontaneous and radiation-induced micronuclei in peripheral blood red cells indicate that POLQ has a role in maintaining genomic integrity via limiting double-strand DNA breaks.
Use of Single Course IV Fludarabine in Treatment of Hypocellular Bone Marrow (Aplastic Anaemia and MDS) Patients.
Authors: Apte Shashikant J. , Kelkar Anjali J. , Melinkeri Sameer , Melinkeri Varsha , Vaidya Sujata , Sayaji Bhavana
Published: October 2019

Young patients with Hypocellular marrow (SAA, VSAA, MDS) ideally need ALLO SCT or Triple Immunosupression with ATG + Cyclosporin + Steroids therapy. However these therapies are very expensive and affordable to very few in third world poor countries. Majority of these patients are treated with androgens with dismal outcome. IV Fludarabine has been used in the setting of ALLO SCT in the preparative regimens and the immunosupressive effect of IV Fludarabine is well established.We decided to assess effect of IV Fludarabine for these patients. The comparative cost is as follows in our country: Allo SCT-$25000/-, ATG+ CYA Therapy- $ 14000/-, IV Fludarabine150mg / m2 - $ 1300/-. At our center we approximately diagnose 75 new cases of SAA, VSAA every year. About 10% undergo ALLOSCT, 15% are treated with ATG+CYA. Approximately 75% patients are treated with androgens. We started this study in patients who could not be treated upfront with standard therapy. W e totally treated 55 patients with SAA, VSAA upfront with IV FLUDARABINE 30mg/m2/day for five successive days. 20 / 55 (36%) patients showed good response and became RBC, Platelet transfusion independant.All these patients achieved ANC > 1500/cmm. 21 / 55 (38%) patients died in first six months. 14 /55 (25%) patients showed no response. Thus SINGLE COURSE IV FLUDARABINE 150 mg /m2 can be a good, cheaper treatment option for newly diagnosed SAA, VSAA.

Patient data Study Period Jan 2001 June 2007 DOSE: IV Fludarabine 30mg / m2 x 5 days Total Patients 55 Age Group 15 – 56 years Males - 37 females -18 Follow up 2 – 54 months Median follow up 36months Haemogram ranges at diagnosis Hb: 3 - 7.4 gm% ANC : 100 – 600cmm Platelet count - 3 -12 x 10′9/l Dysplasia - 12 /55 Cytogenetics 4/55 patients had cytogenetic abnormality : two had del 7 two had del7q 21/55 patients had normal cytogenetics 30/55 patients no metaphases seen on cytogenetics All cellular transfusions were irradiated with 2500 rads. Response 20 / 55 -Response 14 / 55 - No Response 21/55 - Deaths
Expansion of Umbilical Cord Blood Hematopoietic Stem Cells for Clinical Use.
Authors: Peters Rowayda , Hutter Gregor , Aguzzi Adriano , Heikenwaelder Mathias , Renner Christoph , Knuth Alexander
Published: October 2019

Umbilical cord blood (UCB) is an alternative donor source for allogeneic hematopoietic stem cell transplantation. However, transplantation in adults is frequently limited by the small number of cells available in a unit. We have previously developed the technology to expand hematopoietic stem cells (HSC) and stromal/mesenchymal stem cells (SMSC) from all UCB frozen samples (13, 3). The incubation of thawed UCB mononuclear cells (MNC) in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB HSC (non-adherent). Also, long-term expansion of SMSC (adherent cells) was successful. In order to upscale the expansion to use for clinical applications, we analyzed 3 frozen UCB comparing the expansion from 24-well plates (previous) versus expansion in bags (VueLife™) after 10 and 14 days of culture. Results show substantial expansion in total cell count (TCC, 4.8, 10.9: 2.4, 3.8 fold) at d10 and d14 from wells & bags respectively. TCC increased further in the presence of SMSC (38% & 33% in CD34+ cell count cultured in wells). CD133+CD34+CD38- HSC multiplied (11, 25 fold, d10/d14 & colony forming cells (CFC) 19 fold at d14 bags respectively). Heterogeneous cell populations were detected after d 14 in bags: T and B -lymphoid (%CD3/%CD19; 65/4:50w/3), megakaryocytic (%CD61; 7:4) and myeloid (%CD33; 31:43) at d0/d14 respectively. Further, expanded cells (250,000) containing a small number of CD133+CD34+CD38- (15,000–30,000) were injected into the liver of sub-lethally irradiated newborn Balb/C Rag2-Cγ−/− mice. Our preliminary data show no engraftment from cells expanded in wells and bags after 6 weeks of transplantation from d10 cultures (human CD45 + <1%). However, positive engraftment in mouse PB was detected from cells expanded for 14 d (wells, 1.16–2.5% & bags, 1.21–3.9%) as compared to control mice (CD45; 30% PB & 70% BM) receiving selected CD34 + (300,000 CD34+ at d0). Primitive repopulating cells (PRC), and multilineage human CFC were detected after transplantation. On d14 of HSC expansion, UCB MNC derived adherent cells (SMSC) were enriched by trypsinization. SMSC were established in serum free and serum plus culture as well. The immunophenotype of harvested SMSC was CD29+, CD44+ and CD45−, CD34− and CD133− at percentages + >90%. Following repeated trypsinization, SMSC count increased 41–96 folds. CFU-Fibroblast colonies (92–173) were generated from 104 SMSC after 2 weeks in MesenCult medium. We have previously differentiated SMSC into hepatocytes. Now we also generated adipocytes in an induction medium containing, Insulin, dexamethasone & indomethacin. SMSC formed Oil-droplet vacuoles in the cytoplasm in 3 weeks. The culture conditions we defined to maintain UCB PRC should be developed clinically. SMSC described herein exhibit in vitro properties of multipotent stem cells.
Reduction of Prothrombin in Mice Carrying a Conditional Knockout Allele Attenuates the Development of Paralysis Associated with Experimental Encephalitis.
Authors: Mullins Eric S. , Kombrinck Keith W. , Flick Matthew J. , Shaw Maureen A. , Sun William , Degen Sandra J. , Akassoglou Katerina , Degen Jay L.
Published: October 2019

Multiple sclerosis (MS) is common and often devastating chronic inflammatory disease leading to demyelination in the central nervous system and relapsing neurological deficits, including paralysis and vision loss. MS lesions are characterized by blood-brain barrier disruption leading to perivascular deposition of fibrin that correlates with areas of microglia activation and myelin damage. Consistent with multiple studies showing that hemostatic factors can serve as important modulators of the inflammatory response in vivo, we recently identified fibrin(ogen) as a novel regulator of microglial activation/differentiation and showed that fibrinogen plays a causative role in the development of inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE), an established animal model for MS. A working hypothesis that has emerged is that Mac1-mediated microglial cell engagement of fibrin-rich matrices deposited within MS plaques drives activation events leading to neuronal destruction. To better define contribution of the thrombin/fibrin axis in demyelinating neuroinflammatory disease, we challenged conditional “floxed” fII knockout mice carrying low levels (∼10% of normal) of circulation prothrombin (fII) with EAE initiated by immunization with myelin oligodendrocyte peptide. Wild-type mice challenged with EAE typically began to develop overt neurological symptoms within two weeks of immunization. Clinical disease progressed from simple loss of tail tone to ataxia and, ultimately, fore- and hind-limb paralysis. When cohorts were clinically scored in a fashion blinded to animal genotype, prothrombin-deficient mice were found to exhibit significantly reduced disease severity than the control mice tracked in parallel. The timing of disease onset was similar in fII-deficient mice as in controls, consistent with the hypothesis that fII-deficiency diminishes microglial activity. In contrast to control mice that uniformly developed clinically apparent disease, the two-thirds of fII-deficient mice develop either no disease or very mild disease typically limited to the simple loss of tail tone. More detailed studies of fII-deficient mice, including comparative studies of CNS histopathology, are now underway to more fully define the benefits and liabilities of diminished prothrombin on CNS disease. Complementary studies are also underway with gene-targeted mice expressing a mutant form of prothrombin (fII with “specificity switch” favoring protein C over procoagulant substrates). Both prothrombin and fibrinogen are powerful determinants of inflammatory CNS demyelinating disease and a more detailed understanding of the contribution of these factors to disease progression may reveal novel therapeutic strategies for the attenuation of this and other neuroinflammatory diseases.
Relation between FLT3 Mutations, High Flow Cytometric FLT3 Expression and CXCR4 Expression in CD34+ Cells in Adult Acute Myeloid Leukemia.
Authors: Yang Li , Wu Xiao Jin , Wu De Pei
Published: October 2019

Objective

To evaluate the prevalence of FLT3 mutation-interna tanden duplication in adult acute leukemia patients and its clinical significance To evaluate the surface expression of FLT3 difference between AML and non malignant hemopoietic disease, and the possibility of detection of CD135 being included in the diagnostic immunophenotyping of acute leukaemias To study if constitutive FLT3 activity induce cell surface expression of CXCR4 in CD34 cells .

Methods

Genomic DNAs from 95 cases of AML and 22 cases of ALL were screened bypolymerase chain reaction and gel electrophoresis for FLT3-ITD mutations. Flow cytometry is used to investigate FLT3 expression in 68 cases AML and 8cases non-malignant hemopoietic disease To investigate FLT3 expression using flow cytometry between 56 cases FLT3-ITD- AML and 12 cases FLT3-ITD+ AML.

Results

FLT3-ITD were found in 15 (16%) of 95 AL patients,including 3/ 15 of M1, 4/ 24 of M2, 2/ 18 of M3, 5/ 29 of M5, 1/4 of M6.Significantly more FLT3 mututations were found in AML M1, M5. No FLT3-ITD was found in 22 ALL. FLT3-ITD was associated with a higher peripheral white cell count (P<0.05) and a lower complete remission rate (P<0.05).and was more prevalent in patients with normal karyotype(P<0.05) an t(15;17). High level expression of CD135 in acute myeloid leukaemias has been demonstrated by flow cytometry .Dection of CD135 maybe included in the diagnostic immunophenotyping of AML. Significant difference of FLT3 expression between FLT3 ITD posive cases and FLT3 ITD negative cases.
Eltrombopag Decreases Proliferation of Human Leukemia and Lymphoma Cell Lines In Vitro.
Authors: Erickson-Miller Connie L. , Payne Parrish L. , Moore Sherri , Wert Sherryann , May Richard D.
Published: October 2019

Eltrombopag is a novel, oral platelet growth factor that interacts with the thrombopoietin receptor on bone marrow progenitors to stimulate megakaryocyte and platelet production. Clinical trials have demonstrated that eltrombopag increases platelet counts in healthy volunteers and in patients with idiopathic thrombocytopenic purpura (ITP) and hepatitis C. Because thrombopoietin (TPO) receptors are expressed on lymphoma and leukemia cell lines and recombinant human TPO (rhTPO) has been shown to stimulate their proliferation in vitro, experiments were conducted to evaluate the effects of eltrombopag on the proliferation of leukemia and lymphoma cells in vitro. Five leukemia and lymphoma cell lines (ie, CCRF-CEM, K562, MOLT-4, RPMI-8226 and SR) representing lymphoblastic T cell leukemia, chronic myelogenous leukemia, acute lymphoblastic T cell leukemia, plasmacytoma and immunoblastic large cell lymphoma, were exposed to eltrombopag (0.1–40 ug/mL), rhTPO (100 ng/mL) or both for 3 days. Proliferation was measured by 18-hour incubation with tritiated thymidine incorporation. As expected, rhTPO alone led to a small, statistically significant increase in the proliferation of CCRF-CEM and RPMI-8226 cells, while it had no effect on the other cell lines. However, the addition of eltrombopag to rhTPO negated the increased proliferation seen with rhTPO alone. Even more remarkable, treatment with eltrombopag alone inhibited the proliferation of all five leukemia and lymphoma cell lines with an IC50 range of 0.56 to 5.9 ug/mL and 100% inhibition (IC100) of thymidine incorporation at 10 ug/mL (Table). Previous studies have shown that eltrombopag induces proliferation and differentiation of bone marrow progenitor cells in vitro and increases platelet counts in vivo. While eltrombopag and rhTPO interact with the TPO receptor to stimulate the production of platelets, there are differences in the site of receptor interaction and their signaling pathways. The findings of the current study suggest that eltrombopag may inhibit the proliferation of leukemia and lymphoma cell lines unlike the effect that has been demonstrated with rhTPO. Interestingly, eltrombopag may also serve to mitigate rhTPO-mediated proliferation of malignant hematologic cell lines. These findings merit further evaluation of the effects of eltrombopag on leukemia, lymphoma, and solid tumor cell proliferation.

Cell line IC50 (ug/mL) IC100 (ug/mL) CCRF-CEM 0.74 10 K562 1.80 10 MOLT-4 0.56 4 RPMI-8226 5.90 10 SR 0.77 4
Anti-Glycophorin C Induces a Loss of Mitochondrial Membrane Potential but Fails To Activate Apoptotic Caspases.
Authors: Denomme Gregory A. , Micieli Jonathan , Shu Jenny , Wang Dan , Fernandes Bernard J.
Published: October 2019

The human erythrocyte transmembrane sialoglycoprotein, glycophorin C (GYPC), plays a functional role in regulating red cell shape and mechanical stability. Antibodies to GYPC cause hemolytic disease of the fetus and newborn (HDFN) that is associated with classical Fcγ receptor-mediated phagocytosis. However, in vitro clonogenic studies with cord blood progenitor cells suggest that anti-GYPC also suppresses erythropoiesis, which is consistent with the observations of severe and early fetal anemia and late onset neonatal anemia [Transfus Med2005;15:125–32]. The mechanism of the suppressive effect on erythropoiesis is unknown. The K562 erythroleukemic cell line treated with anti-GYPC is a potential model system to study the suppressive effect of anti-GYPC. The present in vitro studies were designed to confirm the effect of anti-GYPC on K562 cell growth and viability, and to evaluate changes in mitochondrial membrane potential, phosphatidylserine (PS) expression, propidium iodide (PI) binding, and caspase activation. K562 cells fail to grow in the presence of anti-GYPC confirming earlier CFU-E/BFU-E studies [Brit J Haematol2006;133:443–4], and increased the exofacial expression of PS/PI over time. This process was caspase-independent as demonstrated by the failure of Z-VAD, a caspase inhibitor, to reverse growth inhibition and PS/PI expression. A loss of mitochondrial membrane potential was demonstrated using JC-1, a cationic dye that is sensitive to potential-dependent accumulation or loss in mitochondria. There was a 50% increase in K562 cell mitochondrial membrane potential disruption after 2 days of culture with anti-GYPC (see figure). Morphological examination of May Grunwalde Giemsa-stained K562 cells treated with anti-GYPC for 2 days showed a decrease in mitotic activity compared to isotype treated cells. By day 4, the anti-GYPC treated cells were showing evidence of plasma membrane damage and cell death resulting from fragmentation and dissolution of the cytoplasm. The addition of hemin, an oxidative form of iron protoporphyrin IX known to induce erythroid differentiation of K562 cells, to anti-GYPC treated cells reversed growth inhibition by 45% but did not prevent the loss of mitochondrial membrane potential. Overall, although caspases appear to be unimportant in anti-GYPC induced cell death, the mitchondria play an important role as the early events leading to antibody-mediated suppression of erythropoiesis.

Mitochondrial Membrane Potential Disruption by Anti-GYPC Mitochondrial Membrane Potential Disruption by Anti-GYPC
The Prolonged Time to Progression with Pegylated Liposomal Doxorubicin + Bortezomib Versus Bortezomib Alone in Relapsed or Refractory Multiple Myeloma Is Unaffected by Extent of Prior Therapy or Previous Anthracycline Exposure.
Authors: Blade Joan , Miguel Jesus San , Nagler Arnon , Sonneveld Pieter , Spencer Andrew , Robak Tadeusz , Hennessey Kathleen , Mundle Suneel D. , Zhuang Sen H. , Harousseau Jean-Luc , Orlowski Robert Z.
Published: October 2019

Recently, a significant improvement in time to progression (TTP) was reported for pegylated liposomal doxorubicin (PLD) + bortezomib combination therapy vs. bortezomib monotherapy in a phase III randomized trial in relapsed or refractory multiple myeloma (RRMM) (Orlowski, JCO 2007). This pre-specified analysis assessed the efficacy of PLD+bortezomib in RRMM based on the number of prior lines of therapy and previous anthracycline exposure status. Patients with ≥1 prior therapy were randomized to receive PLD at 30 mg/m2 on day 4 and bortezomib at 1.3 mg/m2 on days 1, 4, 8, and 11, or bortezomib alone for up to eight 21-day cycles, or at least 2 cycles beyond CR, or optimal response unless disease progression, or unacceptable toxicities occurred. The baseline beta-2 microglobulin levels were comparable between the subgroup with ≥2 prior treatments and that with 1 treatment (median 3.98 vs. 4.01 mg/L), as well as between anthracycline-exposed and -naïve patients (median 3.91 vs. 4.55 mg/L). The improved TTP reported previously with PLD+bortezomib over bortezomib in the total study population was similarly observed in all four subgroups of patients (patients with ≥2 lines of prior therapy or 1 prior therapy, anthracycline-exposed (median 144 mg/m2 in both treatment arms) or -naïve groups (Table)), indicating a consistent therapeutic benefit favoring the PLD+bortezomib combination. Furthermore, TTP was comparable for the PLD+bortezomib combination between groups with ≥2 lines of prior therapy vs. 1 prior therapy (heterogeneity test, p=0.523), as well as patients who were anthracycline-exposed or -naïve (heterogeneity test, p=0.716). Incidence of treatment-related SAEs, grade 3/4 neuropathy, and symptomatic cardiac events was comparable between treatment arms in each subgroup (Table). PLD-related hand-foot syndrome was also <10% in all PLD+bortezomib subgroups. These results demonstrate that PLD+bortezomib combination therapy significantly improves TTP compared to bortezomib alone, regardless of the number of prior lines of therapy, or anthracycline exposure.
Prolonged Overall Survival with Lenalidomide Plus Dexamethasone Compared with Dexamethasone Alone in Patients with Relapsed or Refractory Multiple Myeloma.
Authors: Weber Donna , Knight Robert , Chen Christine , Spencer Andrew , Yu Zhinuan , Zeldis Jerome , Olesnyckyj Marta , Dimopoulos Meletios
Published: October 2019

Introduction: Lenalidomide (Len), an analog of thalidomide (Thal) is a novel, oral, immunomodulatory agent that is effective against multiple myeloma (MM). In 2 prospective, randomized, double-blind, placebo-controlled phase III trials, Len with dexamethasone (Dex) induced a significantly higher overall response (OR) rate and complete remission (CR) rate, as well as longer time-to-progression (TTP) in comparison with Dex alone. Here, we investigate the long-term overall survival (OS) with Len/Dex.

Methods: We evaluated the pooled results from both randomized trials (MM-009, MM-010) of 704 patients who had relapsed or refractory MM, without prior resistance to Dex, who received either Len (25 mg daily for 3 weeks every 4 weeks), or placebo. Dex was given at 40 mg on days 1–4, 9–12, 17–20 every 4 weeks for 4 cycles. From cycle 5 onwards, Dex was given at 40 mg on days 1–4 only. Response rate and TTP are based on data obtained before unblinding (June 2005 [MM-009] and August 2005 [MM-010]). Follow-up data on OS were obtained up to January 2007. Forty-seven percent of patients who received placebo/Dex crossed over to receive Len +/− Dex.

Results: Of 704 patients, 353 were treated with Len/Dex and 351 with Dex alone. Baseline characteristics were well balanced between patients receiving Len/Dex and those receiving Dex alone. Median TTP, OR, and CR were significantly improved in patients treated with Len/Dex compared with Dex alone (Table). Of patients who progressed on Dex alone prior to unblinding, or were found to be receiving Dex alone after unblinding, 47% crossed over to Len +/− Dex. Despite these patients crossing over to Len +/− Dex at progression or at the time of unblinding, the OS was significantly improved in patients treated with Len/Dex compared with Dex alone (hazard ratio 1.295; 95% confidence interval 1.040–1.614; p=0.02). Median OS in the Len/Dex group was 35 months and 31 in the Dex alone group (p<0.05). Median OS was also significantly longer with Len/Dex compared with Dex alone in patients with more than 1 prior therapy (32.4 months versus 27.3 months, p<0.05). Similar median OS was observed with Len/Dex and Dex alone in patients with 1 prior therapy (median OS not yet reached and 35.3 months, p=0.24).

Conclusion: With increased follow-up and despite cross-over, patients treated first with Len/Dex had significantly improved OS compared with those treated with Dex alone.

Len/Dex (n=353) Dex alone (n=351) P value OR, % 60.6 21.9 <0.001 CR, % 15.0 2.0 <0.001 Median TTP, months 11.2 4.7 <0.001 Median OS, months 35.0 31.0 <0.05 Median OS in patients with 1 prior treatment, months not yet reached 35.3 0.24 Median OS in patients with >1 prior treatment, months 32.4 27.3 <0.05
Lineage Shift of a Megakaryocytic-Erythroid Leukemia Cell Line by Adhesion to Fibronectin.
Authors: Yamada Hisashi , Iwase Satsuki , Horiguchi-Yamada Junko
Published: October 2019

Acute megakaryocytic leukemia (AMgL) occurs in about 1% of AMLs and has poor prognosis in adults. Research of AMgL has been hampered by this rarity. To overcome this obstacle, the established immortal AMgL cell lines are useful materials for the study of megakaryocytic malignancies. A few cell lines with the characteristics of megakaryocytes have been established, but half of the cell lines are derived from blastic crisis of CMLs. In normal hematopoiesis, erythroblasts and megakaryocytes have a common precursor. And erythroid and megakaryocytic leukemias frequently share their phenotypes simultaneously. However, the mechanisms of lineage shift between the two remain unknown. We recently established an AMgL cell line named JAS-R. Megakaryocytic-erythroid JAS-R cells showed the phenotypic change by adhesion to culture dishes. Adherent JAS-R cells (named JAS-RAD) show megakaryocytic phenotype concomitant with erythropoietin secretion and MDR1 expression, while suspension cells (JAS-REN) show erythroid characters. Because the adhesion requires bivalent cation and is inhibited by RGDS tetrapeptide, it is presumably mediated by integrins. As for ligands, fibronectin was found to be the most powerful, using dishes coated with collagens (Type I and type IV), laminin, fibronectin and poly-D-lysine. Next we studied the phenotypic changes of JAS-REN cells along with the adhesion to fibronectin. JAS-REN cells were seeded on fibronectin-coated dishes and cultured for 48 hours. Then adherent and floating cells were harvested, separately. Cells were also treated with phorbol ester (TPA). Flow cytometry analysis disclosed that adherent cells had megakaryocytic phenotype with increased CD41a and CD61, and with decreased CD235a. The gene expression profile revealed that FLI1, RUNX1 and GFI1 were increased in fibronectin-adherent cells while GATA1, FOG1 and NFE2 were not changed. These characteristics of fibronectin-adherent cells resembled JAS-RAD. Similar results were observed in TPA-treated cells. Among these transcription factors, the induction of FLI1 was prominent. The promoter activity of FLI1 studied by the luciferase assay was higher in JAS-RAD than JAS-REN. These data suggest the substantial change of AMgL blast cells depending on the growth milieu in vivo. These changes might influence the result of chemotherapy
Methylation Profiling of Apoptotic Genes in Leukemia Cell Lines and Patients.
Authors: EL Ahdab Samih , Castoro Ryan , Abbas Hussein A. , Ahmed Saira , Jelinek Jaroslav , Issa Jean-Pierre
Published: October 2019

^equal contribution Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Apoptosis is executed via 2 main pathways that lead to the activation of caspases: the death receptor (DR) pathway and the mitochondrial pathway. Defects in apoptotic mechanisms play an important role in enabling neoplastic cells to survive by ignoring death inducing signals leading to protection from the lack of survival factors. DNA methylation is a reversible mechanism of gene inactivation that might be responsible for impaired apoptosis in leukemia cells. Methylation profiling of apoptotic genes can identify possible gene targets for epigenetic therapy using demethylating agents like 5-aza-deoxycytidine and azacytidine. It can also be used for classification and prognosis. We performed a comprehensive DNA methylation analysis of apoptosis pathway genes with CpG islands in promoter/exon 1 regions in 5 leukemic cell lines (TF1, K562, Raji, HL60 and Jurkat), 20 patients with acute myelogenous leukemia (AML), 20 patients with acute lymphoblastic leukemia (ALL), and 6 control samples (normal peripheral blood cells). We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Our analysis covered a total of 25 genes: 11 in the intrinsic pathway (BAX, BAK, HRK, CYC1, DIABLO, ATM, APAF1, MAP3K5, CHEK2,PAK1, and BNIP 3), 4 in the extrinsic pathway (TRADD,PYCARD, CARD 10 and BID), 5 genes of the Caspase family (Caspase 2, 3, 6, 7, 9) in addition to 4 of the programmed cell death genes (PDCD 2, 4, 6, and 11). By using a 15% cutoff level to call a gene methylated, methylation was observed in 7 genes in at least one cell line: BNIP3 (Raji), PYCARD (K562), HRK (Tf1, Raji and Jurkat), CARD10 (Raji and HL-60), BID (Raji), PAK1 (Raji) and Casp 6 (Raji). We then performed methylation analysis of 4 genes (BNIP3, PYCARD, PAK1 and CARD 10) in AML and ALL patient samples. Methylation frequencies over a 15% cutoff are summarized in Table 1. We conclude that DNA methylation-associated epigenetic silencing is likely to play a role in modulating apoptosis in a subset of AML and ALL patients, with PYCARD being the most frequent target.

Table 1 Genes Methylation in AML patients Methylation in ALL patients PYCARD 6/20 4/20 BNIP3 1/20 3/20 CARD10 0/20 2/20 PAK1 0/20 1/20
Acetylation of PML Plays a Key Role in Histone Deacetylase Inhibitor-Mediated Apoptosis through Enhanced PML Sumoylation.
Authors: Hayakawa Fumihiko , Kitabayashi Issay , Pandolfi Pier P. , Naoe Tomoki
Published: October 2019

The promyelocytic leukemia (PML) protein is a potent tumor suppressor and proapoptotic factor, and is functionally regulated by posttranslational modification such as phosphorylation, sumoylation, and ubiquitination. Histone deacetylase (HDAC) inhibitors are a promising class of targeted anticancer agents and induce apoptosis to cancer cells. In addition to their effects on histones, HDAC inhibitors increase the acetylation level of several non-histone proteins such as transcription factors, which are important for their effects to cancer cells. However, the mechanism of HDAC inhibitor-induced apoptocis is largely unknown. We report here a novel posttranscriptional modification, acetylation, of PML. By the screening using antibody array, we identified PML as a new acetylation target of Trichostatin A (TSA), a HDAC inhibitor. PML acetylation was enhanced by coexpression of p300 or treatment with TSA. We also showed that increased PML acetylation was associated with increased sumoylation of PML in vitro and in vivo. PML involvement in TSA-induced apoptosis was demonstrated by PML knocking down in Hela cells or PML overexpression in PML−/− MEF cells. Furthermore, PML with acetylation-defective mutation showed disability to mediate the apoptosis, suggesting the importance of PML acetylation for it. Our work provides new insights into the PML regulation by posttranslational modification, and new information about the therapeutic mechanism of HDAC inhibitors.
Human Tissue Factor Pathway Inhibitor-2 Induces Caspase-Mediated Apoptosis in a Human Fibrosarcoma Cell Line (HT-1080).
Authors: Kempaiah Prakasha , Kisiel Walter
Published: October 2019

Human tissue factor pathway inhibitor-2 (TFPI-2) is a 32 kDa extracellular matrix-associated Kunitz-type serine proteinase inhibitor that regulates the plasmin and trypsin- mediated activation of zymogen matrix metalloproteinases and growth factors essential for tumor growth, invasion, and metastasis. We previously demonstrated that HT-1080 human fibrosarcoma cells do not express TFPI-2, but restoration of TFPI-2 expression in these cells by stable transfection with the human TFPI-2 cDNA markedly inhibited their growth and metastasis in-vivo. In this study, 1 μM concentrations of either recombinant human TFPI-2 or its mutated first Kunitz-type domain (R24K KD1), were offered to HT-1080 cells, incubated for 48 h, and the degree of apoptosis assessed by nuclear fragmentation, ethidium bromide/acridine orange (EB/AO) staining, fluorescence-activated cell sorting (FACS) and immunoblotting. Agarose gel electrophoresis of DNA from HT-1080 cells treated with either TFPI-2 or R24K KD1 for 48h indicated DNA fragmentation with a ladder pattern typically associated with apoptosis. EB/AO staining of TFPI-2- and R24K KD1- treated cells revealed that 40–70% of the cells were apoptotic in relation to vehicle-treated cells as judged by fluoresence microscopy. Co-administration of TFPI-2 with polymyxin B produced similar results, suggesting that apoptosis was not endotoxin-dependent. Consistent with our earlier studies showing its enhanced inhibitory activity relative to TFPI-2, R24K KD1 was able to induce apoptosis in 68% of HT-1080 cells after 48h of treatment compared to 39% for the parent TFPI-2 at an equivalent concentration. Moreover, HT-1080 cells treated with a KD1 preparation lacking the reactive site arginine residue (R24Q KD1) produced only an 18% apoptosis rate, thereby linking the observed apoptosis with serine proteinase inhibition. FACS analysis, using propidium iodide and annexin-V labeling, revealed similar apoptotic rates to that seen by EB/AO staining assays. In addition, immunoblotting experiments of vehicle and TFPI-2-treated cells indicated increased caspase-3 activation in TFPI-2-treated cells, thus providing evidence that apoptosis is caspase-mediated. We also observed up-regulation of the proapoptotic Bax protein by immunoblotting following treatment of HT-1080 cells with either TFPI-2 or R24K KD1. Taken together, our results demonstrate that treatment of HT-1080 cells with exogenous TFPI-2 or R24K KD1 activates caspase-mediated, proapoptotic signaling pathways and induces apoptosis. In addition, our data provides suggestive evidence that peritumor application of either TFPI-2 or R24K KD1 may facilitate tumor apoptosis in-vivo.
Sumoylation of TRF1 Is Essential for Its Recruitment to ALT-Associated PML Bodies.
Authors: Yu Jian , Lan Jianping , Zhu Yuanyuan , Lai Xiaoyu , Huang He
Published: October 2019

To achieve unlimited proliferative potential, most cancer cells activate telomerase to maintain telomeres. However, some cancer cells elongate telomeres through a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). These ALT cells contain a novel promyelocytic leukemia (PML) body (ALT-associated PML body, APB), which comprises telomeric DNA and a number of proteins, including PML protein, the telomere binding proteins TRF1 and TRF2, replication factor A, and recombination factors Rad51, Rad52, and the Rad50/Mre11/NBS1 complex. TRF1, as the first identified telomeric binding protein, binds the duplex telomeric repeats at telomere ends. It plays an important role in telomere length control, telomeric ends shelter and cell cycle regulation. In ALT cell lines, TRF1 co-localized with PML protein at APBs, but the exact mechanism of its recruitment to APBs is not clear. Here we show that TRF1 localizes to PML bodies in about 5% of an asynchronously growing culture of U2OS cells and the percentage of cells with colocalization of TRF1 and PML bodies increases to about 40% in cells enriched in G2/M, which is consistent with the previous studies. Furthermore, our results show that TRF1 is modified by the small ubiquitin-like protein SUMO-1 in vivo and in vitro. Firstly, 293T cells were transfected with Flag-TRF1, HA-Ubc9, GFP-SUMO1 and then immunoprecipitated by using FLAG-M2 gel under denaturing conditions. Immunoblotting with GFP and Flag antibodies demonstrated that TRF1 is modified by SUMO-1 in vivo. Next, in vitro SUMO-1 conjugation assay of TRF1 was employed. The results showed that TRF1 is conjugated with SUMO-1 in the presence of purified recombinant protein SAE1/SAE2, Ubc9, SUMO1, His-TRF1 and ATP. Either SAE1/SAE2, Ubc9, SUMO-1 or ATP was omitted from the reaction abolished the sumoylation of TRF1. Previous studies have shown that sumoylation controls the recruitment of several proteins to PML bodies, so we examined whether sumoylation of TRF1 is required for its recruitment to APBs. We mutated the potential sumoylation sites of TRF1 according to the computational prediction and then transfected it into U2OS cells to examine its localization. The results showed that TRF1 mutant does not localize to PML bodies. Taken together, all these results suggest that TRF is modified by SUMO-1 and sumoylation of TRF1 is essential for its recruitment to APBs in ALT cells.
The Kinase Inhibitors Sunitinib (Sutent®) and Sorafenib (Nexavar®) Differentially Affect Reactivity of NK Cells Against Renal Cell Cancer.
Authors: Manuela Schlicke , Matthias Krusch , Baessler Tina , Kanz Lothar , Salih Helmut R.
Published: October 2019

Sunitinib (Sutent®) and Sorafenib (Nexavar®) are multikinase inhibitors licensed for the treatment of patients with advanced renal cell cancer (RCC) because of their direct and indirect anti-tumor and anti-angiogenic effects. Sunitinib and Sorafenib target, among others, vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), the stem cell factor receptor (cKIT) and Fms-like tyrosine kinase-3 (Flt3). Beyond that, Sorafenib additionally inhibits c-RAF and B-RAF. NK cells play an important role in the immune surveillance of tumors. Their reactivity is governed by different surface receptors leading to activation of various kinases, phosphatases and transcription factors. Up to now, nothing is known regarding the influence of Sunitinib and Sorafenib on NK cells. Here we report that Sunitinib and Sorafenib differentially affect NK cell cytotoxicity. Analysis of peripheral blood mononuclear cells from 10 independent donors revealed that Sorafenib but not Sunitinib significantly (p<0.05, Mann-Whitney U-test) reduced cell surface CD107a expression (about 50%) on NK cells as a surrogate marker for degranulation in response to tumor targets. In line, pharmacological levels of Sorafenib significantly (p<0.05, Mann-Whitney U-test) diminished cytotoxicity of primary NK cells against the RCC cell lines A498, ACHN and CAKI-2 (from 46% to 14%, from 52% to 22% and from 63% to 42%, respectively) in chromium release assays. Again, no inhibition of cytotoxicity was observed in the presence of pharmacological concentrations of Sunitinib. Since activation of MAP kinases leading to ERK phosphorylation regulates the movement of lytic granula towards target cells, which is responsible for NK cell cytotoxicity we investigated the influence of Sunitinib and Sorafenib on ERK phosphorylation. While Sunitinib had no effect, Sorafenib, alike the specific c-RAF inhibitor ZM336372 or the specific MEK inhibitor PD98059, markedly reduced ERK phosphorylation as determined by Western Blot. Our results indicate that NK cell anti-tumor reactivity is impaired by Sorafenib but not by Sunitinib due to the additional inhibitory effect of Sorafenib on the ERK pathway, which has been demonstrated to dictate NK cell cytotoxicity. We therefore conclude that Sunitinib is exquisitely suitable for combination with immunotherapeutic approaches in RCC patients.
Morphine Stimulates the Phosphorylation and Nuclear Translocation of EGFR in Lung Cancer Cells.
Authors: Nguyen Julia , Johnson Katherine H.N. , Pasrija Teena , Kratzke Robert A. , Hyams Aaron R. , Stinson Brittany A. , Gupta Kalpna
Published: October 2019

Mu opioid receptor (MOR) stimulates MAPK/ERK phosphorylation, by transactivating epidermal growth factor receptor (EGFR) in astrocytes (Belcheva MM et al J Biol Chem 2001, 276, 33847–53) and by transactivating vascular endothelial growth factor receptor-2 (VEGFR2) in endothelial cells (Chen C et al Curr Neurovasc Res 2006, 3, 171–80). EGFR and VEGFR2 are two critical targets for lung cancer therapy. Since opioids used to treat pain in cancer act via MOR, it is critical to understand the role of MOR in growth and survival promoting signaling in lung cancer. Therefore, we examined if MOR co-activates, EGFR mediated signaling in non-small cell human lung cancer cells, HL2009, HL1299 and HL460; and VEGFR2 in murine blood outgrowth endothelial cells (BOEC) derived from wild type (wt) and MOR knockout (KO) mice. We observed that HL2009 cells showed a 3–5 fold higher expression of MOR (by RT-PCR) as well as constitutive activation of phospho-EGFR (by Western) as compared to HL1299 and HL460. Morphine (100 nM) stimulated the phosphorylation of EGFR, MAPK/ERK and Akt in a time-dependent manner similar to that induced by 10 nM EGF, in HL2009 but not in HL460 cells. Morphine as well as EGF-induced phosphorylation peaked after 10 and 30 min of stimulation and it was accompanied by the translocation of phospho-EGFR to the nucleus in HL2009 but not in HL460 cells. Since, nuclear expression of phospho-EGFR is associated with poor prognosis in some human cancers, it is possible that MOR activation upon opioid exposure may impart resistance to lung cancer therapy, by stimulating the translocation of phospho-EGFR to the nucleus. On the other hand, morphine also stimulated the phosphorylation of VEGFR2 and MAPK/ERK in BOEC from wt mice but not in BOEC from MOR KO mice. Thus, opioid analgesics may stimulate VEGFR2 via MOR and stimulate angiogenesis. Acquired resistance to EGFR therapy in non small cell lung cancer is associated with increased VEGF activity. Therefore, simultaneous co-activation of EGFR and VEGFR2 via MOR in lung cancer may promote cancer progression and resistance to VEGF and/or EGFR based therapies. We speculate that MOR-specific antagonists in combination with VEGF/EGFR based therapies may prove to be beneficial in treating lung cancer.
Poor Survival of CD34 Positive and CD13/33 Positive Patients in Pediatric T-Cell Acute Lymphoblastic Leukemia Is Not Explained by Expression of Multidrug Resistance Genes.
Authors: van Grotel Martine , van Noesel Max M. , Van Den Heuvel M.M. , Pieters Rob , Meijerink Jules P.P.
Published: October 2019

For childhood T-ALL, the significance of early differentiation markers CD34 and CD13/33 in relation to outcome is unclear. In this study of 72 pediatric T-ALL patients, CD34 and CD13/33 expression was related to a poor 5 yrs survival (p=0.0013 for CD34+ and p=0.035 for CD33/CD13+). Expression of CD34 was associated with elevated MDR1 (p=0.01) and MRP1 (p=0.019) levels and CD13/33+ expression with MRP1 levels (p=0.028). However, none of these MDR genes independently predicted for poor outcome. We conclude that T-ALL with CD34 and CD13/33 expression has a relatively poor survival that is not explained by expression of MDR genes.
Immunoglobulin and T-Cell Receptor (Ig/TCR) Gene Rearrangements in Chinese Adult Acute Lymphoblastic Leukemia Patients.
Authors: Yao Li , Chen Zi-xing , Cen Jian-nong , He Jun , Qi Xiao-fei , Shen Hong-jie
Published: October 2019

To determine the feasibility of Ig/TCR gene rearrangements as patient-specific targets for the detection of minimal residual disease in Chinese ALL patients, forty adult patients with B-lineage and T cell acute lymphoblastic leukemia(ALL) were enrolled in this study. The frequency and pattern of IGH, IGK, TCRB, TCRG and TCRD gene rearrangement were analyzed by BIOMED-2 multiplex PCR protocol. All DNA samples were obtained from the bone marrow of these patients. IGH, IGK, TCRB, TCRG and TCRD rearrangements were found in 86%,14%,27%,77%,59% of adult patients with B-lineage ALL, respectively. While in T-ALL, clonal TCRB, TCRG and TCRD rearrangements were detected in 83%,78%,39% of patients. There are several features in the pattern of Ig/TCR gene rearrangements in Chinese adult ALL patients. Firstly, IGK rearrangement in adult B-lineage ALL had a lower frequency than in children’s pre-B-ALL (14% vs 41%) in reported literature. Secondly, the frequency of TCRG Vrfl-Jr1.3, Vrfl-Jr1.1 and Vr9-Jr1.3 rearrangement was 65%,6%,29% in adult patients with B-lineage ALL, while 60%,27%,13% was the frequency of those in T-ALL, respectively. In conclusion, our data have shown that several special patterns in Ig/TCR gene rearrangements in Chinese adult ALL patients, may provide a guidance for individual treatment protocol designation and MRD-based risk stratification.
Molecular Monitoring of ERG and BAALC as Minimal Residual Disease Markers in Patients with Acute Leukemia.
Authors: Najima Yuho , Ohashi Kazuteru , Kakihana Kazuhiko , Kikuchi Taku , Nagata Yasunobu , Sakurai Chihiro , Ueki Toshimitsu , Kobayashi Takeshi , Yamashita Takuya , Kawamura Machiko , Akiyama Hideki , Kaku Hidefumi , Sakamaki Hisashi
Published: October 2019

Although expression of the genes ERG (ETS-related gene) and BAALC (brain and acute leukemia, cytoplasmic) predicts outcome in acute leukemia, there is little information about their utility as markers of minimal residual disease (MRD). A real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) was used to examine relative levels of ERG and BAALC gene expression in 11 patients with acute leukemia. Pretreatment and post-chemotherapy bone marrow (BM) samples were analyzed to quantify these transcripts. Peripheral blood (PB) samples were also obtained to evaluate the correlation of ERG and BAALC expression levels in BM and PB samples. Significant levels of ERG gene were expressed in all patients. In contrast with ERG, the levels of BAALC gene expression were significantly lower in some patients. Continuous monitoring of the ERG mRNA was performed and assessed a correlation between MRD levels and subsequent clinical courses. In patients with relative high expression of BAALC transcripts (>7x10exp-3/GAPDH), high levels of ERG gene expressions were consistently observed whether they were in remission status or not. In 2 patients with acute promyelocytic leukemia (APL), MRD was simultaneously monitored by RQ-PCR using primers for PML/RAR alpha. Changes in expression levels of PML/RAR alpha and ERG presented poor correlation. While, in other patients with relative low expression of BAALC transcripts (<7x10exp-3/GAPDH), ERG gene expression levels gradually decreased after the induction therapy and following consolidation treatments. In patients unable to attain remission, ERG expression levels remained high. Thus, the quantitation of the ERG gene expression made it possible to assess MRD in patients with acute leukemia (except for APL) carrying low BAALC expression. To our knowledge, this is the first report concerning the use of ERG mRNA expression for MRD monitoring.