In Vitro Studies of Bortezomib with Daunorubicin and Cytarabine:Sequence of Administration Affects Leukemia Cell Chemosensitivity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4464-4464 ◽  
Author(s):  
Eyal C. Attar ◽  
Emily Learner ◽  
Philip C. Amrein
Keyword(s):  
Cell Cycle ◽  
Proteasome Inhibitor ◽  
Remission Rate ◽  
Single Agent ◽  
Leukemia Cell ◽  
In Vitro Studies ◽  
Leukemia Cell Line ◽  
Sequential Administration ◽  
Synergistic Cytotoxicity

Abstract Induction chemotherapy with cytarabine and an anthracycline has been a standard treatment of newly diagnosed AML for the past 20 years. While the complete remission rate is high, only 20–40% of patients have prolonged leukemia-free survival and thus novel treatments are required. Bortezomib represents a first-in class proteasome inhibitor with activity in a variety of hematologic malignancies. Recent evidence has demonstrated synergistic cytotoxicity between the proteasome inhibitor, MG-132, and the anthracycline, idarubicin, in primitive leukemia cells.(Guzman et al. Proc Natl Acad Sci U S A 99 2002 16220-5) In order to determine if adding bortezomib to the conventional antileukemic therapies daunorubicin and cytarabine enhances cellular cytotoxicity, we conducted in vitro studies on the acute myeloid leukemia cell line, KG-1. Proliferation assays demonstrated that single-agent daunorubicin or single-agent cytarabine impaired proliferation to a greater extent than when either agent was simultaneously combined with bortezomib. Reasoning that treatment with bortezomib resulted in cell cycle arrest and protection from cycle-dependent agents, we treated cells with bortezomib and observed an accumulation of cells in the G2/M phase of the cell cycle, supporting our hypothesis. We then sought to determine whether sequential administration of bortezomib and chemotherapy affected leukemia cell proliferation. Cells were treated with bortezomib either 24 hours before or after idarubicin or cytarabine. Surprisingly, pretreatment of cells with bortezomib followed by chemotherapy resulted in increased proliferation relative to chemotherapy alone while initial treatment of cells with chemotherapy followed by bortezomib resulted in enhanced cytotoxicity. In conclusion, these data indicate that in in vitro leukemia cell cultures the timing of administration of bortezomib relative to conventional anti-leukemic agents critically affects cytotoxicity.

scholarly journals Tumor Inhibiting [1,2-Bis(fluorophenyl)ethylenediamine]platinum(II) Complexes Part II: Biological Evaluation -in vitro Studies on the P 388 D1 Leukemia Cell Line

1990 ◽  
Vol 323 (3) ◽  
pp. 133-140 ◽  
Author(s):  
Herta Reile ◽  
Richard Müller ◽  
Ronald Gust ◽  
Reiner Laske ◽  
Walter Krischke ◽  
...  
Keyword(s):  
Cell Line ◽  
Leukemia Cell ◽  
Biological Evaluation ◽  
In Vitro Studies ◽  
Leukemia Cell Line

Addition of Bortezomib (Velcade) to AML Induction Chemotherapy Is Well Tolerated and Results in a High Complete Remission Rate.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2782-2782 ◽  
Author(s):  
Eyal C. Attar ◽  
Daniel J. De Angelo ◽  
Andres Sirulnik ◽  
Martha Wadleigh ◽  
Karen K. Ballen ◽  
...  
Keyword(s):  
Complete Remission ◽  
Proteasome Inhibitor ◽  
Remission Rate ◽  
Performance Status ◽  
Complete Recovery ◽  
Prior History ◽  
Group Setting ◽  
Ecog Performance Status ◽  
Synergistic Cytotoxicity

Abstract In vitro experiments have shown that the addition of a proteasome inhibitor to an anthracycline results in synergistic cytotoxicity to leukemia cells and, specifically, the leukemia stem cell. Hence, we initiated this phase I study to determine if the proteasome inhibitor, bortezomib, could be safely added to conventional treatment for acute myeloid leukemia (AML). Eligibility was restricted to patients ≥ 18 with relapsed disease after a remission of at least 3 months and patients ≥ 60 without prior treatment for AML. All patients were required to have an ECOG performance status of 0–3 and adequate cardiac, renal, and hepatic function. Patients with ≥ grade 2 peripheral neuropathy prior to enrollment were excluded. All patients received idarubicin 12 mg/m2 on days 1–3 and cytarabine 100 mg/m2 by continuous infusion days 1–7. Bortezomib was added to this regimen on days 1, 4, 8, and 11 by IV bolus. Cohorts of 3–6 patients were treated with the following doses of bortezomib: 0.7, 1.0, 1.3, and 1.5 mg/m2. An additional 6 patients were to be treated at the candidate maximally tolerated dose (MTD). Dose limiting toxicities (DLTs) were defined as prolonged myelosuppression, severe neuropathy, and other grade 3 or 4 toxicities. Dose escalation was permitted if < 2 DLTs were experienced in 3–6 patients at a given dose level. The study is now closed, with 30 patients entered and 24 patients evaluable at this time. The age range was 42–75 years, with a median of 65. There were 13 males and 11 females. Of these patients, 16 were ≥ 60 with previously untreated AML, of whom 4 had a prior history of MDS or MPD, 4 were ≥ 60 with relapsed AML, and 4 < 60 with relapsed AML. Among the first 6 patients treated at 0.7 mg/m2, there was 1 DLT consisting of prolonged neutropenia. In the second cohort of 6 patients treated with 1.0 mg/m2 bortezomib, there was 1 DLT consisting of prolonged thrombocytopenia. No DLTs were encountered in 9 patients treated with 1.3 mg/m2 bortezomib. Because the MTD had not been reached, an additional cohort assessing 1.5 mg/m2 bortezomib was added. Three patients have completed treatment at this dose and no DLTs were experienced; a final 6 patients are currently undergoing treatment. There have been no significant neurologic or cardiac toxicities. There was one death within the first 45 days of protocol treatment. This occurred in the setting of febrile neutropenia in a patient previously transplanted for AML who received 1.5 mg/m2 bortezomib. Of the first 24 patients, 14 (58%) achieved complete remission (CR), 3 achieved remission without complete recovery of platelet count (CRp), 3 achieved a partial remission (6–24% BM blasts), and 4 patients failed to respond. In conclusion, bortezomib was well tolerated up to 1.3 mg/m2 in this regimen and this combination produced an encouraging remission frequency in this population of patients. A phase II study of this combination will proceed in the cooperative group setting.

Lenalidomide Inhibits Proliferation of Chronic Lymphocytic Leukemia Cells in Vitro

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1775-1775
Author(s):  
Jessie-Farah Fecteau ◽  
Diahnn Futalan ◽  
Ila Bharati ◽  
Emanuela M. Ghia ◽  
Laura G Corral ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Research Funding ◽  
Single Agent ◽  
Leukemia Cell ◽  
S Phase ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Cell Expression

Abstract Abstract 1775 Promising clinical responses have been observed in chronic lymphocytic leukemia (CLL) patients treated with lenalidomide as a single agent or in combination with other agents, The mechanisms of action of lenalidomide are under study; unlike most other anti-leukemia drugs, lenalidomide has no direct cytoxic effects in vitro on primary CLL cells, which typically are in G0/1 phase of the cell cycle. We examined the activity of lenalidomide on CLL cells that were induced to proliferate in vitro. To induce proliferation, CLL cells were cultured in media containing human interleukin (IL)-4 and IL-10 and with stromal cells (HeLa) made to express CD154. Labeling CLL cells with carboxyfluorescein succinimidyl ester (CFSE), allowed us to monitor for several rounds of induced CLL-cell division via flow cytometry. We found that addition of 0.33–10 micro M lenalidomide to such cultures resulted in a dose-dependent reduction in the number of leukemia cells induced to undergo cell-division. Moreover, we found that lenalidomide could significantly reduce the number of dividing CLL cells in each patient sample tested (n=4) by an average of 1.7 fold, reducing the fraction of dividing cells from 77% ± 27% to 44% ± 22% after 6 days of culture (mean +/− SD, P < 0.05). Evaluation of the DNA content of CLL cells using propidium iodide (PI) and flow cytometry revealed that lenalidomide significantly decreased the percentage of CLL cells in the G2/M phase of the cell cycle from 9% ± 2.7% to 5.1% ± 2.1% (mean +/− SD, n=4; P< 0.05) in control versus lenalidomide-treated cultures, respectively. Furthermore, lenalidomide appeared also to reduce the percentages of CLL cells in S phase from 12% ± 8% to 6.2% ± 3.1%. We found that the capacity of lenalidomide to inhibit CLL cell-division was associated with lenalidomide-induced leukemia-cell expression of p21/WAF/Cip, which can directly inhibit the activity of cyclin-dependent kinases required for progression from G1 into the S phase of the cell cycle. Gene expression analysis of CLL cells (n=10) revealed that lenalidomide induced increased leukemia-cell expression of p21/WAF/Cip at 6h and at 24h, an effect that also was noted for leukemia cells in the blood of patients treated with single-agent lenalidomide. Lenalidomide-induced expression of p21/WAF/Cip was associated with induced expression of the pro-apoptotic protein Bim, a downstream target of of p21/WAF/Cip. However, lenalidomide did not appear to induce leukemia-cell expression of TP53, which can induce p21/WAF/Cip, suggesting that lenalidomide induces p21/WAF/Cip via a TP53-independent mechanism. These results suggest that lenalidomide has effects on CLL cells that are distinct from those induced by CD40-ligation. Moreover, these studies reveal a potential mechanism for the anti-leukemia activity of lenalidomide, which might inhibit factors that potentially drive leukemia-cell proliferation in vivo. Disclosures: Corral: Celgene: Employment. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding. Messmer:Celgene: Research Funding.

Molecular Characterization Of The Anti-Leukemic Effects Of Single Agent Eltrombopag

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4923-4923
Author(s):  
Uwe Platzbecker ◽  
Katja Sockel ◽  
Claudia Schönefeldt ◽  
Daniel Nowak ◽  
Susann Helas ◽  
...  
Keyword(s):  
Complete Remission ◽  
Disease Progression ◽  
Cell Line ◽  
Cell Lines ◽  
Copy Number ◽  
Single Agent ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Dose Dependent

Abstract Introduction Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist which has been shown in-vitro to inhibit leukemia cell growth. The underlying mechanism is still under investigation. Methods We report a patient with NPM1 mutated/FLT3 negative refractory AML who achieved a complete remission during treatment with single agent EP within the PMA112509 trial. In this patient we conducted sequential molecular analyses out of the bone marrow to study the underlying molecular mechanisms. Therefore, samples prior to EP, at remission and relapse were subjected to genome-wide copy number analysis using Affymetrix SNP 6.0 array in search for acquired copy number alterations (CNA). To screen for alterations in commonly mutated genes in AML, samples were further subjected to a next generation deep sequencing assay (NGS) of mutational hotspots in the genes ASXL1, CBL, DNMT3A, ETV6, EZH2, IDH1/2, KRAS, NPM1, NRAS, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. Sequencing was performed on the 454 GS Junior platform (Roche applied science). Moreover we investigated the expression of TPO-R (CD110) by different assays in cell lines and primary AML samples. To study the TPO-R dependency of potential antineoplastic EP effects we studied the effects of lentiviral TPO-R knockdown and single agent EP on the vitality and cell cycling of TPO-expressing and non-expression leukemia cell lines. Results By using NGS we followed the NPM1+ mutation (NPM1 c.864incTCTG) load in this patient and found a concomitant decline (prior EP: 12.6%, at CR: 1.1%) but not disappearance of NPM1+ cells and a reemergence (15.2%) together with a clonal evolution and development of a NRAS c.37G>C mutation during disease progression (Figure 1) while a SNP-array demonstrated no additional CNA at disease progression. Real time PCR analysis demonstrated TPO-R expression at all time points analyzed which declined during complete remission(TPO-R/GAPDH: prior EP: 56.7%, at CR: 32.3%). These results prompted us to study TPO-R expression of blasts by flow cytometry in de novo AML samples (n=72) at diagnosis. In fact, TPO-R was expressed only in 33 of 72 AML patients but across all FAB and cytogenetic subgroups. The median MFI (mean fluorescence intensity) of CD110 was 2-fold higher on blasts than on CD110 positive lymphocytes. Interestingly, there were some differences with regards to the mutational status, since the NPM mutation was documented more frequently in CD110 negative than in CD110 positive AML cases (26% vs. 10%). These data were confirmed by Taqman-PCR in an independent cohort (n=57) with a nearly three fold lower expression of TPO-R on NPM1+/FLT3- compared to NPM1-/FLT3- (p=0.0163) cases. Next, we sought to clarify if TPO-R expressing AML cell lines are dependent on TPO-R expression. Knockdown of TPO-R by lentivirally transferred shRNA resulted in down-regulation and rapid cell death in the TPO-R expressing megakaryoblastic cell line (CMK). However, treatment with EP in-vitro at doses ranging from 1 to 10 µg/ml lead to a dose-dependent decrease in the cell division rate and vitality not only in CMK but also in cell-lines with weak or absent surface TPO-R expression (e.g. KG1a, a human acute myeloid leukemia cell line or OCI-AML3, a NPM1+ myeloid cell line). In parallel, a significant counterregulatory upregulation of TPO-R mRNA was observed which was dose-dependent (KG1a, p=0.0014). Conclusion These data demonstrate that TPO-R is heterogeneously expressed across all AML subtypes but absent in the majority of NPM1+/FLT3- cases. The clinical response seen in our patient with a refractory NPM1+ AML further provides evidence to the fact that single agent EP can exert potent anti-leukemic effects in-vivo. These effects seem to be mediated rather independently of TPO-R expression. Disclosures: Platzbecker: GlaxoSmithKline: Honoraria, Research Funding.

Thyroid hormone responsiveness of the L1210 murine leukemia cell line

1992 ◽  
Vol 126 (4) ◽  
pp. 374-377 ◽  
Author(s):  
J Brtko ◽  
P Filipčík ◽  
J Knopp ◽  
V Sedláková ◽  
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Protein Phosphorylation ◽  
Nuclear Receptors ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
L1210 Leukemia ◽  
Murine Leukemia Cell ◽  
Murine Leukemia

The presence of saturable and high affinity 3,5,3′-triiodothyronine (T3) binding sites was demonstrated in LI 210 murine leukemia cell nuclei. Scatchard analysis revealed one class of receptors for T3 with Ka = 2.187 × 109l/mol and a maximum binding capacity (Bmax) of 3.96 fmol/106 cells. The effects of T3 on protein phosphorylation and growth rate of L1210 cells were investigated in a medium containing T3-depleted fetal calf serum. T3 was observed to be effective in enhancing protein phosphorylation (153.06%±5.99 sd) compared to cells grown in the absence of T3 (81.49%±13.50 sd). Moreover, in the presence of high T3 concentration (11.15 nmol/l) T3 was found to significantly increase the cell growth rate. In addition, the T3 receptor-associated alterations during the cell cycle, as measured by flow cytometry, suggest that the presence of T3 receptors becomes evident during the late G3 phase of the cell cycle, and T3 receptor numbers increase during the S phase. These results suggest that in in vitro conditions representing high T3 concentration, the number of L1210 leukemia cells may be increased by T3 via nuclear receptors. The L1210 leukemia cell line may serve as a convenient tool for in vitro studies of nuclear receptors and/or mechanism of action of T3. The binding affinity of T3 receptors is similar to that found in rat hepatocytes or human lymphocytes.

Reversal of Methotrexate-Induced Folate Pool Depletion by Thymidine in a Human Leukemia Cell Line in Vitro

1993 ◽  
Vol 79 (6) ◽  
pp. 433-438 ◽  
Author(s):  
Pratima Sur ◽  
Yoshinobu Matsuo ◽  
Takeshi Otanl ◽  
Jun Minowada
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Folate Level ◽  
Ifn Γ

In an In vitro study using a human monocytic leukemia cell line, U-937, the effects of interferon-γ (IFN-γ) in combination with the antifolate methotrexate and the role of thymidine introduced as a biochemical modulator were investigated. Methotrexate alone or in combination with INF-γ was found to enhance the induction of morphologic and functional monocytic differentiation in the U-937 cell line. Various cellular effects with the addition of thymidine to the medium with methotrexate and IFN-γ were studied. Enhanced inhibition of cell growth and perturbation of the cell cycle were noted when methotrexate and IFN-γ were used in combination, but not when methotrexate was used alone. The reduction of cellular folate by methotrexate was also enhanced in combination with IFN-γ. Cell cycle delay, resulting in cell growth inhibition of folate depletion, caused the induction of differentiation in U-937 cells, which was found to be greater with methotrexate + IFN-γ than with methotrexate alone. Cellular differentiation, as assessed by nitroblue tetrazolium reduction assay, indirect immunofluorescence and morphology, showed better effects towards the differentiation of U-937 cells when the agents were used in combination. However, addition of thymidine to the medium was found to cancel all the aforementioned effects. The addition of thymidine to the medium also caused reversal of the inhibitory effect of methotrexate and IFN-γ on cell growth and repletion of the endogenous folate level. Repletion of the folate level by exogenous thymidine is a new possibility for the role of the thymidine in cellular growth.

CDK1-mediated phosphorylation of the RIIα regulatory subunit of PKA works as a molecular switch that promotes dissociation of RIIα from centrosomes at mitosis

2001 ◽  
Vol 114 (18) ◽  
pp. 3243-3254 ◽  
Author(s):  
Cathrine R. Carlson ◽  
Oliwia Witczak ◽  
Lutz Vossebein ◽  
Jean-Claude Labbé ◽  
Bjørn S. Skålhegg ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Regulatory Subunit ◽  
Wild Type ◽  
Soluble Extract ◽  
Stable Transfectants ◽  
Anchoring Protein ◽  
Cell Fractions

Protein kinase A regulatory subunit RIIα is tightly bound to centrosomal structures during interphase through interaction with the A-kinase anchoring protein AKAP450, but dissociates and redistributes from centrosomes at mitosis. The cyclin B-p34cdc2 kinase (CDK1) has been shown to phosphorylate RIIα on T54 and this has been proposed to alter the subcellular localization of RIIα. We have made stable transfectants from an RIIα-deficient leukemia cell line (Reh) that expresses either wild-type or mutant RIIα (RIIα(T54E)). When expressed, RIIα detaches from centrosomes at mitosis and dissociates from its centrosomal location in purified nucleus-centrosome complexes by incubation with CDK1 in vitro. By contrast, centrosomal RIIα(T54E) is not redistributed at mitosis, remains mostly associated with centrosomes during all phases of the cell cycle and cannot be solubilized by CDK1 in vitro. Furthermore, RIIα is solubilized from particular cell fractions and changes affinity for AKAP450 in the presence of CDK1. D and V mutations of T54 also reduce affinity for the N-terminal RII-binding domain of AKAP450, whereas small neutral residues do not change affinity detected by surface plasmon resonance. In addition, only RIIα(T54E) interacts with AKAP450 in a RIPA-soluble extract from mitotic cells. Finally, microtubule repolymerization from mitotic centrosomes of the RIIα(T54E) transfectant is poorer and occurs at a lower frequency than that of RIIα transfectants. Our results suggest that T54 phosphorylation of RIIα by CDK1 might serve to regulate the centrosomal association of PKA during the cell cycle.

scholarly journals Facile synthesis of Au/ZnO/Ag nanoparticles using Glechoma hederacea L. extract, and their activity against leukemia

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Renata Dobrucka ◽  
Aleksandra Romaniuk-Drapała ◽  
Mariusz Kaczmarek
Keyword(s):  
Electron Microscopy ◽  
Ag Nanoparticles ◽  
Mononuclear Cells ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Mtt Test ◽  
Transmission Electron ◽  
Almost All ◽  
Glechoma Hederacea

AbstractMetal combinations have been attracting the attention of scientists for some time. They usually exhibit new characteristics that are different from the ones possessed by their components. In this work, Au/ZnO/Ag nanoparticles were synthesized biologically using Glechoma hederacea L. extract. The synthesized Au/ZnO/Ag nanoparticles were characterized by UV-Vis, Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), and Atomic Force Microscopy (AFM). The microscopic methods confirmed the presence of spherical nanoparticles of 50–70 nm. The influence of biologically synthesized Au/ZnO/Ag nanoparticles on the vitality of human cells was evaluated in vitro with the use of established human Acute T Cell Leukemia cell line, Jurkat (ATCC® TIB-152™), as well as mononuclear cells isolated from peripheral blood (PBMC) of voluntary donors. Cell survival and the half-maximal inhibitory concentration index (IC50) were analyzed by the MTT test. The studies showed that the total loss of cell viability occurred at the Au/ZnO/Ag nanoparticle concentration range of 10 µmol–50 µmol. The use of Au/ZnO/Ag nanoparticles at the concentration of 100 µmol eliminated almost all living cells from the culture in 24h. The above observation confirms the result obtained during the MTT test.

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