scholarly journals Correlation of P-glycoprotein expression and function in childhood acute leukemia: a children's cancer group study

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 309-318
Author(s):  
SP Ivy ◽  
RS Olshefski ◽  
BJ Taylor ◽  
KM Patel ◽  
GH Reaman
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Childhood Leukemia ◽  
Alkylating Agents ◽  
Chemotherapeutic Agents ◽  
Myelogenous Leukemia ◽  
Response To Treatment ◽  
Acute Leukemias ◽  
P Glycoprotein ◽  
And Function

Clinical drug resistance may be attributed to the simultaneous selection and expression of genes modulating the uptake and metabolism of chemotherapeutic agents. P-glycoprotein (P-gp) functions as a membrane-associated drug efflux pump whose increased expression results in resistance to anthracyclines, epipodophyllotoxins, vinca alkaloids, and some alkylating agents. This type of resistance occurs as both de novo and acquired resistance to therapy for leukemia. We have studied P- gp expression and function in childhood acute leukemias by developing a series of doxorubicin- and vincristine-selected CEM, T-cell lymphoblastoid cell lines that recapitulate the low levels of expression and resistance seen clinically. These cell lines have been used to develop flow cytometric assays for the semiquantitative measurements of P-gp expression with the MRK16 monoclonal antibody and P-gp function using the enhanced retention of rhodamine 123 in the presence of verapamil, a resistance modulator. Kolmogorov-Smirnov statistics, represented by the D measurement, are used to determine the difference in level of P-gp expression by comparing MRK16 staining to an IgG2a isotype control. When D is > 0.09, there is an excellent correlation (R = 0.82) between P-gp expression and function. The evaluation of 107 bone marrow specimens from 84 children with lymphoblastic or myelogenous leukemia showed a statistically significant (P = .004) increase in P-gp function at relapse. P-gp expression at relapse, however, approached but did not reach a significant level (P = .097). Using this methodology, we can identify patients with levels of P-gp expression and function that we can define clinically, as well as children with discordant multidrug resistance phenotypes. This study supports the role of P-gp-mediated drug resistance in childhood leukemia and confirms that P-gp expression and function are measurable in their leukemic blasts. These assays provide the means for the in vitro testing of resistance modulators and the monitoring of in vivo response to treatment with these agents.

scholarly journals Correlation of P-glycoprotein expression and function in childhood acute leukemia: a children's cancer group study

Blood ◽  
1996 ◽  
Vol 88 (1) ◽  
pp. 309-318 ◽  
Author(s):  
SP Ivy ◽  
RS Olshefski ◽  
BJ Taylor ◽  
KM Patel ◽  
GH Reaman
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Childhood Leukemia ◽  
Alkylating Agents ◽  
Chemotherapeutic Agents ◽  
Myelogenous Leukemia ◽  
Response To Treatment ◽  
Acute Leukemias ◽  
P Glycoprotein ◽  
And Function

Abstract Clinical drug resistance may be attributed to the simultaneous selection and expression of genes modulating the uptake and metabolism of chemotherapeutic agents. P-glycoprotein (P-gp) functions as a membrane-associated drug efflux pump whose increased expression results in resistance to anthracyclines, epipodophyllotoxins, vinca alkaloids, and some alkylating agents. This type of resistance occurs as both de novo and acquired resistance to therapy for leukemia. We have studied P- gp expression and function in childhood acute leukemias by developing a series of doxorubicin- and vincristine-selected CEM, T-cell lymphoblastoid cell lines that recapitulate the low levels of expression and resistance seen clinically. These cell lines have been used to develop flow cytometric assays for the semiquantitative measurements of P-gp expression with the MRK16 monoclonal antibody and P-gp function using the enhanced retention of rhodamine 123 in the presence of verapamil, a resistance modulator. Kolmogorov-Smirnov statistics, represented by the D measurement, are used to determine the difference in level of P-gp expression by comparing MRK16 staining to an IgG2a isotype control. When D is > 0.09, there is an excellent correlation (R = 0.82) between P-gp expression and function. The evaluation of 107 bone marrow specimens from 84 children with lymphoblastic or myelogenous leukemia showed a statistically significant (P = .004) increase in P-gp function at relapse. P-gp expression at relapse, however, approached but did not reach a significant level (P = .097). Using this methodology, we can identify patients with levels of P-gp expression and function that we can define clinically, as well as children with discordant multidrug resistance phenotypes. This study supports the role of P-gp-mediated drug resistance in childhood leukemia and confirms that P-gp expression and function are measurable in their leukemic blasts. These assays provide the means for the in vitro testing of resistance modulators and the monitoring of in vivo response to treatment with these agents.

scholarly journals Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Birthe Gericke ◽  
Saskia Borsdorf ◽  
Inka Wienböker ◽  
Andreas Noack ◽  
Sandra Noack ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Blood Brain Barrier ◽  
Cell Lines ◽  
Chemotherapeutic Agents ◽  
Brain Barrier ◽  
Brain Capillaries ◽  
Brain Capillary ◽  
P Glycoprotein ◽  
Blood Brain ◽  
And Function

Abstract Background In vitro models based on brain capillary endothelial cells (BCECs) are among the most versatile tools in blood–brain barrier research for testing drug penetration into the brain and how this is affected by efflux transporters such as P-glycoprotein (Pgp). However, compared to freshly isolated brain capillaries or primary BCECs, the expression of Pgp in immortalized BCEC lines is markedly lower, which prompted us previously to transduce the widely used human BCEC line hCMEC/D3 with a doxycycline-inducible MDR1-EGFP fusion plasmid. The EGFP-labeled Pgp in these cells allows studying the localization and trafficking of the transporter and how these processes are affected by drug exposure. Here we used this strategy for the rat BCEC line RBE4 and performed a face-to-face comparison of RBE4 and hCMEC/D3 wild-type (WT) and MDR1-EGFP transduced cells. Methods MDR1-EGFP-transduced variants were derived from WT cells by lentiviral transduction, using an MDR1-linker-EGFP vector. Localization, trafficking, and function of Pgp were compared in WT and MDR1-EGFP transduced cell lines. Primary cultures of rat BCECs and freshly isolated rat brain capillaries were used for comparison. Results All cells exhibited typical BCEC morphology. However, significant differences were observed in the localization of Pgp in that RBE4-MDR1-EGFP cells expressed Pgp primarily at the plasma membrane, whereas in hCMEC/D3 cells, the Pgp-EGFP fusion protein was visible both at the plasma membrane and in endolysosomal vesicles. Exposure to doxorubicin increased the number of Pgp-EGFP-positive endolysosomes, indicating a lysosomotropic effect. Furthermore, lysosomal trapping of doxorubicin was observed, likely contributing to the protection of the cell nucleus from damage. In cocultures of WT and MDR1-EGFP transduced cells, intercellular Pgp-EGFP trafficking was observed in RBE4 cells as previously reported for hCMEC/D3 cells. Compared to WT cells, the MDR1-EGFP transduced cells exhibited a significantly higher expression and function of Pgp. However, the junctional tightness of WT and MDR1-EGFP transduced RBE4 and hCMEC/D3 cells was markedly lower than that of primary BCECs, excluding the use of the cell lines for studying vectorial drug transport. Conclusions The present data indicate that MDR1-EGFP transduced RBE4 cells are an interesting tool to study the biogenesis of lysosomes and Pgp-mediated lysosomal drug trapping in response to chemotherapeutic agents and other compounds at the level of the blood–brain barrier.

scholarly journals Changes in IDH2, TET2 and KDM2B Gene Expression After Treatment With Classic Chemotherapeutic Agents and Decitabine in Myelogenous Leukemia Cell Lines

2019 ◽  
Vol 8 (3) ◽  
pp. 89-101
Author(s):  
Jayse Alves ◽  
Georgia Muccillo Dexheimer ◽  
Laura Reckzigel ◽  
Marcia Goettert ◽  
Vanderlei Biolchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Chemotherapeutic Agents ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Lines

Minimal Residual Disease in Hematologic Disorders

1999 ◽  
Vol 123 (11) ◽  
pp. 1030-1034
Author(s):  
Stefan Faderl ◽  
Razelle Kurzrock ◽  
Zeev Estrov
Keyword(s):  
Minimal Residual Disease ◽  
Residual Disease ◽  
Chemotherapeutic Agents ◽  
New Drugs ◽  
Myelogenous Leukemia ◽  
Therapeutic Success ◽  
Acute Leukemias ◽  
Hematologic Disorders ◽  
Kinetics Of ◽  
Minimal Residual

Abstract In almost no other area of medical oncology has the introduction of new drugs, combinations of chemotherapeutic agents, and novel biologic treatments caused such dramatic responses as it has in the treatment of malignant hematologic disorders. However, despite some therapeutic success, many patients relapse and die from recurrence of their disease. The implications of minimal residual disease (MRD), a term referring to disease that is undetectable by conventional morphologic methods, have therefore attracted increasing attention in recent years. New and powerful laboratory tools such as polymerase chain reaction assays have extraordinary sensitivity and provide exciting new insights into the detection, nature, quantification, and kinetics of MRD. This article summarizes methods used in the identification of MRD and its importance as exemplified in the case of acute leukemias and chronic myelogenous leukemia.

Expression of Cyclooxygenase-2 (COX-2) in Human Leukemias and Hematopoietic Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4336-4336 ◽  
Author(s):  
Michael B. Lilly ◽  
Leslie Drapiza ◽  
Milan Sheth ◽  
Marina Zemskova ◽  
Svetlana Bashkirova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Growth Factor ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Myelogenous Leukemia ◽  
Human Leukemia ◽  
Flow Cytometry Assay ◽  
Acute Leukemias ◽  
Cox 2 ◽  
Cox 2 Inhibitors

Abstract COX-2 has been implicated in the development of many epithelial cancers, as well as in tumor angiogenesis. COX-2 inhibitors have been shown to have anti-tumor activity in experimental cancer. Little information exists, however, on the expression or role of COX-2 in hematologic malignancies. We have use a variety of immunochemical assays to document expression of COX-2 in human and murine leukemias and hematopoietic cells. The factor-dependent murine cell lines FDCP1 and 32D expressed COX-2 when growing continuously in the presence of IL-3; expression declined markedly when growth factor was removed. FDCP1 cells constitutively expressing bcl-2, pim-1, or bcr-abl had markedly elevated levels of COX-2, and continued to express this enzyme even after removal of growth factor. To assess COX-2 expression in human hematopoietic cells we developed a flow cytometry assay using a FITC-labelled anti-COX-2 MoAb (Cayman). Cells were washed once in serum-free medium, fixed briefly in 1% paraformaldehyde, permeabilized with PBS/0.2% Triton X100, then stained with antibody. Negative control samples were processed similarly but stained with antibody that had been preincubated with immunizing peptide. Specific COX-2 staining was interpreted as the difference between the histograms from blocked versus unblocked anti-COX-2 antibody, as determined by Kolmogorov-Smirnoff analysis. In buffy coat preparations from normal donors, we found constitutive expression of COX-2 in lymphocytes (both B-cells and T-cells). In contrast little or no COX-2 was detected in unstimulated neutrophils or monocytes. In human acute myelogenous leukemia (AML) cell lines we found COX-2 expression to be universal and easily detected. In several cell lines we confirmed the results of our flow cytometry assay with immunoblotting. We further examined 25 cryopreserved samples of human acute leukemia blasts obtained from peripheral blood. COX-2 expression was variable, but universal. Levels generally were less than those seen in immortalized cell lines, and did not correlate with blasts morphology (AML, ALL, APL, AMoL, CML-BT). To determine if COX-2 inhibitors could play a role in the treatment of acute leukemias, we performed cytotoxicity assays using the COX-2 specific inhibitors, celecoxib and NS398. Survival and growth of human AML cell lines were inhibited by both agents. These data demonstrate that 1) a variety of oncogenes can induce expression of COX-2 in hematopoietic cells, 2) clinical human acute leukemias uniformly express COX-2 in circulating blasts, and 3) COX-2 inhibitors are cytotoxic for human leukemia cells. Combination therapies for acute leukemias may evaluate the incorporation of COX-2 inhibitors for added cytotoxic effects or angiogenesis inhibition.

Combination of a Spicamycin Analogue KRN5500 and Chemotherapeutic Agents Synergistically Enhances Their Cytotoxicity in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4783-4783
Author(s):  
Hirokazu Miki ◽  
Shuji Ozaki ◽  
Osamu Tanaka ◽  
Shiro Fujii ◽  
Shingen Nakamura ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Growth ◽  
Cell Lines ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Human Tumor ◽  
Chemotherapeutic Agents ◽  
Dependent Manner ◽  
Induced Apoptosis ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. Although new classes of agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains an incurable disease due to increased resistance to these agents. Therefore, alternative approaches are necessary to overcome drug resistance in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan). This drug potently decreases protein synthesis and inhibits cell growth in human tumor cell lines both in vitro and in vivo. Several phase I studies of KRN5500 were conducted in patients with solid tumors, which showed Cmax values of 1000–3000 nM at the maximum tolerated doses. However, no objective anti-tumor response to KRN5500 alone was observed in these patients. In this study, we examined the anti-tumor activity of KRN5500 against MM cells and evaluated its therapeutic potential in combination with other anti-MM agents. MM cell lines and freshly-isolated MM cells were incubated with various concentrations of KRN5500 for 24 hours. Cell proliferation assay showed marked inhibition of cell growth in MM cells such as RPMI 8226, KMS12-BM, and UTMC-2 (IC50 = 10–40 nM), and U266, MM.1S, and primary MM cells (IC50 = 500–1000 nM). Importantly, a chemotherapy-resistant subclone of RPMI 8226 had a similar sensitivity to KRN5500. Annexin V/propidium iodide staining confirmed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Moreover, cleavage of poly (ADP-ribose) polymerase (PARP) was detected after 24 hours with only modest activation of caspase-8, -9, and -3 by immunoblotting. Flow cytometric analysis of anti-apoptotic proteins revealed that apoptosis induced by KRN5500 was associated with down-regulation of Mcl-1 and Bcl-2 expression. To determine the effect of KRN5500 on the unfolded protein response (UPR), splicing of XBP-1 mRNA was analyzed by reverse transcription-polymerase chain reaction. In response to stimulation with KRN5500, splicing of XBP-1 mRNA occurred after 24 hours in RPMI 8226 cells, suggesting that KRN5500-induced apoptosis is mediated in part by the inhibition of UPR. Furthermore, synergistic effects on MM cells were observed when KRN5500 was combined with anti-MM agents including melphalan, dexamethasone, and bortezomib. These results suggest that KRN5500 induces apoptosis in MM cells mainly by the caspase-independent pathway and that its unique mechanism of action provides a valuable therapeutic option to overcome drug resistance in patients with MM.

HSP70 Inhibitor, YK5, Synergizes with Chemotherapeutic Agents and Prevents Chemoresistance in Acute Myelogenous Leukemia (AML).

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2476-2476
Author(s):  
Krishan K. Sharma ◽  
Juan Felipe Rico ◽  
Michael W. Becker ◽  
Gail J. Roboz ◽  
Gabriela Chiosis ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Resistance ◽  
Cell Death ◽  
Heat Shock ◽  
Quantitative Pcr ◽  
Caspase 3 ◽  
Chemotherapeutic Agents ◽  
Fold Change ◽  
Myelogenous Leukemia ◽  
Intracellular Hsp70

Abstract Abstract 2476 Stress-inducible heat shock protein 70 (HSP70) is a major cytoprotective factor and a molecular chaperone that interacts with HSP90 to form a multi-chaperone complex. Cancer cells are highly dependent on this complex due to their increased demand for protein synthesis. HSP70 overexpression inhibits apoptosis and has been associated with drug resistance and poor prognosis. YK5, a novel inhibitor of tumor-HSP70, has been shown to induce potent cell death in AML blast, progenitor, and stem cell populations with minimal effects in normal hematopoietic cells. Due to the role of HSP70 in drug resistance, we examined the effect of combining YK5 with other chemotherapeutic agents, including arsenic trioxide, cytarabine, suberoylanilide hydroxamic acid (SAHA) and PU-H71, a novel tumor-specific HSP90 inhibitor. We tested the ability of YK5 to synergize with either AsO3, AraC, SAHA, or PU-H71 in primary AML samples. Using multiparameter flow cytometry to measure viability after 48 hours of treatment, we found that combining 1μM YK5 with either 500nM AsO3 or PU-H71 resulted in a significant increase in cell death when compared to either agent alone (n=9, mean viability: 51.8, 67.2, and 13.4% for AsO3, YK5, and AsO3/YK5, respectively, P = 0.0018; mean viability: 57.1 and 20.8% for PU-H71 and PU-H71/YK5, respectively, P = 0.0029). A synergistic relationship between YK5 and both AsO3 and PU-H71 was found in all nine primary samples (combination indexes 0.29 – 0.76 with YK5/AsO3, 0.33 – 0.83 with YK5/PU-H71). In contrast, the combination of YK5 with either AsO3 or PU-H71 in CD34+ cord blood mononuclear cells did not result in a significant increase in cell death when compared to either agent alone (mean viability: 42.4, 72.4, and 37.2% for AsO3, YK5, and AsO3/YK5, respectively; mean viability: 61.1 and 51.1% for PU-H71 and PU-H71/YK5, respectively). YK5 in combination with either AraC or SAHA, however, did not result in a significant increase in cell death when compared to either drug alone, with an additive effect being demonstrated with a 1:1 YK5 to AraC/SAHA drug ratio (Mean CI = 0.9918). To determine the mechanism of the observed synergistic activity, intracellular HSP70 and active caspase-3, a client of HSP70, were measured using flow cytometry. Both AsO3 and PU-H71 significantly increased intracellular HSP70 and caspase-3 (Mean fold change = 18.3, 21.0 of HSP70 and 9.9, 8.3 of Caspase-3 for AsO3 and PU-H71 treatment, respectively), while treatment with AraC or SAHA resulted in no change in HSP70 levels. Furthermore, quantitative PCR revealed that treatment with either AsO3 or PU-H71 strongly upregulated HSPA1A and HSPA6, the main stress-inducible isoforms of HSP70 (Mean fold change = 15.9, 14.1 of HSPA1A, and 20.8, 23.4 of HSPA6 for AsO3 and PU-H71 treatment, respectively). AraC and SAHA had no significant upregulation of these genes. We have previously shown that increased levels of HSPA1A correlate with sensitivity to HSP70 inhibition via YK5. To further explore the mechanism of this observed synergy, flow cytometry was used to measure the levels of reactive oxygen species (ROS). Treatment with AsO3, PU-H71, AraC, or SAHA resulted in a significant increase in ROS (Mean fold change = 2.75, 1.92, 2.89, 1.67, respectively). Quantitative PCR also confirmed the activation of the oxidative stress response by the upregulation of heme oxygenase 1 (HMOX1) by treatment with these drugs (Mean fold change = 10.9, 8.7, 11.2, 7.7, respectively). YK5, however, did not induce ROS or upregulate HMOX1. Interestingly, pretreatment with NAC in primary AML samples (n=4) resulted in no protection from YK5 synergistic effect when combined with either AsO3 or PU-H71. These results suggest that YK5 synergizes with AsO3 and PU-H71 due to the increase in intracellular HSP70 caused by these drugs. This synergy is most likely due to the activation of the heat shock response and independent of the production of ROS due to drug treatment. In summary, we have found that the novel tumor-HSP70 inhibitor YK5 can synergize with AsO3 and PU-H71 in primary human AML, and that the basis of this synergism is due to the increase in intracellular HSP70 caused by these chemotherapeutic agents. HSP70 inhibition represents a novel approach in AML treatment and can be particularly significant to drug-resistant patients when combined with other chemotherapy. Disclosures: Roboz: Astex Pharmaceuticals: Research Funding.

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