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NEOPLASIA
From the Department of Interdisciplinary Oncology and
Clinical Investigations Program and the Biostatistical Department, H. Lee Moffitt Cancer Center and Research Institute, University of South
Florida, Tampa, FL.
We previously showed that adhesion of myeloma cells to fibronectin
(FN) by means of Studies have found that cellular adhesion by means
of Topo II is an adenosine triphosphate (ATP)-dependent enzyme that
reversibly cuts double-stranded DNA and is transiently linked to the 5'
end of the break site by phosphotyrosyl bonds. Mammalian cells contain
2 isoforms of topo II (topo II Cell culture
Drugs and antibodies
Cell-surface expression and functional adhesion assay Cell-surface expression of integrins was measured by incubating 1 million cells with primary or isotype control antibody for 30 minutes on ice.1 After 2 washes with phosphate-buffered saline (PBS), cells were incubated with a secondary fluorescein isothiocyanate (FITC)-conjugated goat antimouse antibody (Dako). After incubation with the secondary antibody, the samples were washed twice with PBS. Fluorescence was analyzed by flow cytometry using a fluorescence-activated cell-sorter scanner (Becton Dickinson, Mountain View, CA) to record 10 000 events. Mean fluorescence values for the isotype control were subtracted from the mean fluorescence values for integrin staining. Mean and SD values were calculated from the results of 3 independent experiments.The adhesion assay was done as described previously.1
Briefly, 96-well Immunosorp (Nunc, Denmark) plates were coated with either 50 µL (40 µg/mL) of soluble FN (Gibco) or bovine serum albumin (BSA) and allowed to evaporate overnight at room temperature. Cells were washed once in serum-free RPMI medium and resuspended at a
density of 1 × 106 cells/mL. Before cell adhesion, cells
were incubated for 30 minutes with a 1:100 dilution of either isotype
control,
Inhibition of cell growth Inhibition of cell growth was determined by using a modified monotetrazolium (MTT) dye assay with the following modifications.1 A 96-well Immunosorp plate was coated with FN as described previously.1 Briefly, cells were washed once in serum-free RPMI medium, and FN samples were plated at a density of 150 000 cells/mL. Cells in suspension were incubated in serum-free RPMI medium in a conical tube at the same density as cells attached to FN. After 2 hours of cellular adhesion, wells were aspirated and 180 µL RPMI medium containing 10% FCS was added to each well. Cells maintained in suspension were centrifuged and resuspended in RPMI medium containing 10% FCS at a concentration of 100 000 cells/mL. Plates were treated with various concentrations of drug for 1 hour. After 1 hour of drug exposure, plates were washed 3 times with RPMI containing 10% FCS. After a 72-hour incubation at 37°C, 50 µL MTT dye (2 mg/mL) was added to each well, and the cells were incubated for an additional 4 hours. Plates were centrifuged once at 500g, medium was aspirated, the water-insoluble product was dissolved in DMSO, and absorbance was read at 490 nm on an automatic plate reader. The concentration of drug that produced 50% inhibition of growth (IC50) was calculated by using linear regression analysis.Apoptosis A flow cytometric assay assessing annexin V staining was used to count apoptotic cells after drug exposure, as described previously.1 After 2 hours of adhesion to FN, cells were exposed to drug for 1 hour and extracellular drug was removed by 3 washes with RPMI medium containing 10% FCS. For experiments examining etoposide-induced apoptosis, cells were exposed continuously to various doses of etoposide. Apoptotic cells were detected 20 hours after initial drug exposure by using annexin V staining and flow cytometric analysis. Ten thousand events were analyzed by flow cytometry (Becton Dickinson, San Jose, CA). Mean and SD values were calculated from the results of at least 3 independent experiments done in duplicate. Statistical comparisons used the Student t test.Comet assay Cells were placed in serum-free RPMI medium (750 000 cells/mL) and either adhered to FN-coated, 35-mm plates (Nunc) or placed in suspension for 2 hours. After 2 hours of adhesion, cells were exposed to various concentrations of drug for 1 hour. Subsequently, 5000 cells were placed in a microcentrifuge tube containing 1 mL cold PBS, and the neutral comet assay was done as described by Kent et al.12 Briefly, cells were centrifuged and resuspended in 500 µL cold PBS, and 1.5 mL 1% agarose was added to each sample. The agarose-cell suspension was gently layered on a frosted-glass microscope slide, allowed to solidify for 5 minutes, and then placed immediately in ice-cold lysis buffer containing 30 mM disodium ethylenediamine tetraacetic acid (EDTA, pH 8.0), 0.5% sodium dodecyl sulfate (SDS), and 0.25 mg/mL proteinase K (Fisher Scientific, Norcross, GA). The samples were lysed for 1 hour at 4°C and then kept at 37°C for 12 to 16 hours. After cell lysis and digestion of protein-DNA complexes with proteinase K, the agar slides were re-equilibrated in TBE (90 mM Tris-hydrochloric acid, 90 mM boric acid, and 2 mM EDTA [pH 8.0]) for 2 hours, with a change of buffer every 15 minutes. The samples were electrophoresed with TBE buffer for 20 minutes at 25 V. The DNA was then stained with a 1:10 000 dilution of Sybr Green (Molecular Probes, Eugene, OR) for 20 minutes and slides were washed twice for 5 minutes in TBE. To ensure random sampling, 50 images/slide were captured and, in some experiments, the observer was blinded to the conditions. The images were captured on a fluorescent microscope (Vysis, Downers Grove, IL) and quantified by using Imagequant software (Molecular Dynamics, Sunnyvale, Ca). The comet moment was calculated by using the following equation described by Kent et al12: comet moment = 0 n ((intensity of DNA at distance
X) × (distance))/intensity of total DNA.
The mean comet-moment value obtained from vehicle-control samples was subtracted from the mean comet-moment value for each drug dosage. Data shown are the mean and SD values from 3 independent experiments (50 images for each dose of each independent experiment). An analysis of variance (ANOVA) model was used to quantify the relation between the response variable and the 2 independent variables. The response variable in the analysis was the difference between the mean comet-moment values for the control and drug-treated samples. Independent variables were the dosage of mitoxantrone (0.1 µM, 1 µM, and 10 µM) and the treatment type (FN versus suspension). The variance estimate for the test statistic was calculated by pooling the variances from each of the 2 groups (control and treated). Accumulation of carbon 14 (14C)-mitoxantrone Cellular accumulation of 14C-mitoxantrone in FN-adhered cells and cells in suspension (1 × 106) was compared after a 1-hour exposure to 2.5 µM 14C-mitoxantrone (specific activity, 0.3 GBq/mM). After exposure to drug at 37°C for 1 hour, FN-adhered and suspension cells were washed 3 times in cold PBS. The cells were counted before cell lysis with 10% SDS, and the data were normalized to counts per minute of 14C-mitoxantrone/1 million cells.Topo II activity and expression Cells in log-phase growth were washed once in serum-free RPMI medium, resuspended at density of 1 × 106 cells/mL in serum-free RPMI medium, and adhered to FN or placed in suspension as described previously.1 Nuclear extracts were prepared as described by Sullivan et al.7 All the following procedures were done at 4°C, and 1 mM phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, and 5 µg/mL aprotinin were added to buffers B to F. For FN samples, cells remained on FN-coated plates until placed in a dounce homogenizer. Approximately 25 million cells were washed once in PBS and then washed once with 25 mL buffer A (0.15 M sodium chloride [NaCl] and 10 mM potassium phosphate, monobasic [KH2PO4]). Samples were incubated for 30 minutes on ice with 4 mL buffer B (5 mM KH2PO4, 2 mM magnesium chloride [MgCl2], 4 mM dithiothreitol [DTT], and 0.1 mM sodium (NA2) EDTA). Cells were then dounce homogenized for 15 strokes; the release of nuclei was followed microscopically before proceeding to the next step. Nuclei were collected at 2500g for 15 minutes, further purified by resuspension in 2 mL buffer C (buffer B and 0.25 M sucrose), and layered over 1 mL buffer D (buffer B and 0.6 M sucrose). The sucrose gradient was centrifuged in a swinging-bucket rotor for 20 minutes at 2000g. The nuclear pellet was resuspended in 100 µL buffer E (5 mM KH2PO4, 4 mM DTT, and 1 mM Na2 EDTA), and the total volume was measured. An equal volume of buffer F (40 mM Tris [pH 7.5], 2 M NaCl, and 4 mM DTT) was added to the solution, which was incubated for an additional 30 minutes on ice. The lysate was centrifuged at 100 000g for 1 hour, and the supernatant was adjusted to 10% glycerol (vol/vol). To decrease the chance of topo II degradation, all topo activity assays were done on the same day the nuclear extracts were obtained.For immunoblotting, 30 µg fresh nuclear extract from suspension and
FN-adhered samples were separated on a 7.5% SDS-polyacrylamide gel and
transferred to a polyvinylidene difluoride membrane. The blot was
probed with either a topo II The catalytic activity of topo II was measured as the decatenation of networks of kinetoplast DNA (kDNA). The kDNA was labeled with tritium-thymidine and isolated from Crithidia fasciculata as described previously.7 One microgram nuclear protein extract and 0.40 µg kDNA was incubated in a total volume of 40 µL at 30°C for selected times. The reaction buffer consisted of the following: 50 mM Tris (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1.0 mM ATP, 0.5 mM DTT, and 30 µg/mL BSA. The reaction was terminated by the addition of 5 µL 2.5% SDS. The samples were then microcentrifuged for 10 minutes at 12 000 rpm at room temperature. After centrifugation, 30 µL of the supernatant, which contained the released kDNA minicircles, was removed, liquid scintillation fluid was added, and radioactivity was measured with a scintillation counter (Beckman, Palo Alto, CA). Immunohistochemical analysis Confocal microscopy was used to determine whether cellular adhesion changed the intracellular localization of topo II. U937 cells were adhered to FN for 2 hours. After 2 hours of cellular adhesion, cells maintained in suspension or cells adhered to FN were fixed with 4% paraformaldehyde for 10 minutes. The cells were then cytospinned and subsequently permeabilized with 0.5% Triton-X, 1% glycine, and PBS for 1 hour as described previously.10 Briefly, after permeabilization, slides were incubated with either a 1:100 dilution of topo II or topo II polyclonal antibody for 1 hour (0.1% NP-40
and 1% BSA in PBS). After several washes in PBS for 2 hours,
slides were incubated with a goat anti-rabbit immunoglobulin G
(IgG)-tetrarhodamine isothiocyanate-labeled antibody (Sigma) at a
1:80 dilution in 0.1% NP-40 and 1% BSA in PBS for 35 minutes in the
dark. After incubation with the secondary antibody, slides were washed
several times in PBS for 2 hours. Immunofluorescence was observed with
a scanning confocal microscope (LSM 510; Zeiss, Göttingen,
Germany). To obtain nuclear-to-cytoplasmic ratios of FN-adhered cells
and cells in suspension, 100 individual cells were analyzed for pixel
density of the nucleus and cytosol. The mean pixel density of the
background was subtracted from all values before calculation of the
nuclear-to-cytoplasmic ratio.
Adhesion to FN is mediated by 4, 5, and 1 integrin subunits is shown in Figure
1A. The U937 cells expressed more 4
(mean fluorescence, 91.32 ± 31.74) than 5 (mean fluorescence,
36.11 ± 15.97); however, the use of blocking antibodies showed
that adhesion of U937 cells is mediated primarily by 51 integrin
(Figure 1B).
Adhesion of U937 cells to FN for 2 hours causes resistance to topo II inhibitors The MTT assay was used to determine whether the adhesion of U937 cells to FN protects cells from drug-induced cytotoxicity. As shown in Figure 2, adhesion of U937 cells to FN for 2 hours before drug exposure increased the IC50 value of mitoxantrone approximately 10 fold (range, 5-17 fold). The IC50 value of doxorubicin was increased approximately 3 fold (range, 2-5 fold). The degree of resistance for the nonintercalating topo II inhibitor etoposide, as measured by MTT assay, was less than that for mitoxantrone and doxorubicin, being approximately 2 fold (range, 1.3-4 fold).
In addition to measuring cytotoxicity with the MTT assay, we used an
apoptosis assay to assess the effects of FN adhesion on drug-induced
apoptosis. FITC-conjugated annexin V, which binds to inverted
phosphatidylserine on the surface of the plasma membrane, was used to
identify apoptotic cells. As shown in Figure
3A, cells adhered to FN before a 1-hour
exposure to various doses of mitoxantrone had reduced apoptosis on
annexin V staining. Cells treated while adhered to FN were also
protected from etoposide-induced apoptosis (Figure 3B). These data
indicate that adhesion of U937 cells to FN protects against
mitoxantrone-induced apoptosis and, to a lesser extent,
etoposide-induced apoptosis. Studies using doxorubicin were not done
because the drug interfered with this fluorescence assay.
Adhesion of U937 cells to FN reduces mitoxantrone- and etoposide-induced DNA double-strand breaks as measured by neutral comet assay Mitoxantrone and etoposide are known to stabilize topo II-DNA complexes, resulting in DNA double-strand breaks.13-15 We used the neutral comet assay to compare the amount of mitoxantrone- or etoposide-induced DNA double-strand breaks in U937 cells that were either exposed to drug in suspension or adhered to FN. The comet moment is a function of both the distance and the amount (measured in pixel density) of DNA that migrates from the center of the head of the comet. As shown in Figure 4, the tail length, tail intensity, and tail shape differed according to whether the cells were treated with drug in suspension or while adhered to FN. After 2 hours of adhesion to FN, both mitoxantrone- and etoposide-induced comet-moment values were decreased by approximately 40% to 60% compared with results in cells treated in suspension (Figure 4E-F). The ANOVA showed a significant difference between cells treated with drug in suspension and adhered to FN (P < .01 for all doses tested).
A drug-accumulation assay was done to determine whether the reduction in drug-induced DNA double-strand breaks correlated with reduced intracellular drug accumulation. Total intracellular 14C-mitoxantrone was measured after a drug exposure of 1 hour, a time consistent with the measurement of drug-induced DNA damage. The adhesion-dependent decrease in DNA double-strand breaks could not be attributed to decreased intracellular drug accumulation (counts per minute for 14C-mitoxantrone, 8853 ± 1329 in cells in suspension and 10 266 ± 1052 in FN-adhered cells; no significant difference at the P < .05 level). These data indicate that the reduction in cytotoxicity and DNA damage that occurs when cells are adhered to FN is not due to a decrease in intracellular concentration of the drug. These findings are consistent with our previous study showing that adhesion of the multiple myeloma 8226 cell line to FN did not alter the intracellular concentration of doxorubicin.1 Adhesion of U937 cells to FN reduces salt-extractable topo II
activity and topo II levels were decreased by 66% (mean value from 3 independent experiments) in cells adhered to FN compared with cells
grown in suspension. In contrast, adhesion of cells to FN did not alter
the nuclear levels of topo II (Figure 5C). Furthermore, topo I
levels remained constant in nuclear extracts prepared from either cells
in suspension or FN-adhered cells (Figure 5D).
Confocal microscopy was used to determine whether cellular adhesion to
FN altered the cellular distribution of topo II
To assess whether cellular adhesion to FN altered the nuclear binding
properties of topo II
Durand and Sutherland18 were among the first
investigators to show that changes in the microenvironment can alter
the response to radiation. In their model, cells grown as a spheroid
were more resistant to drugs and radiation than cells grown as a
monolayer. The increase in drug resistance associated with spheroid
cultures compared with monolayer cultures was shown to correlate with
decreased drug-induced DNA damage.19 These results suggest
that cell-cell contact or cell-matrix contact modulates the cellular
response to drug-induced DNA damage. Moreover, the decrease in
drug-induced DNA damage was found to be correlated with the
redistribution of topo II We previously showed that In this study, we found that adhesion of U937 cells to FN by means of
Topo II is the putative target of mitoxantrone and etoposide that
causes DNA double-strand breaks. Thus, qualitative or quantitative changes in topo II can decrease such drug-induced breaks. We observed that adhesion of U937 cells to FN resulted in diminished topo II
catalytic activity as measured by the release of minicircles from kDNA.
The decrease in enzymatic activity correlated with a decrease in
salt-extractable nuclear topo II Topo II has been shown to be posttranscriptionally modified by
phosphorylation and ribosylation,23,24 and perhaps
posttranscriptional modifications alter the affinity of topo II There is evidence that etoposide and mitoxantrone inhibit both topo
II Little is known about extracellular signals that regulate expression
and activity of topo II In summary, we found that adhesion of U937 cells to FN attenuates DNA
double-strand breaks induced by mitoxantrone and etoposide. The
decrease in these breaks correlated with a decrease in topo II
catalytic activity and salt-extractable topo II
We thank the H. Lee Moffitt Analytical Microscopy Core Facility, the H. Lee Moffitt Flow Cytometry Core Facility, the H. Lee Moffitt Biostatistics Core Facility, all of which are supported by National Institutes of Health grant P30-CA76292-04-08; and Peggy Farrell for careful editing of the manuscript.
Submitted December 18, 2000; accepted May 10, 2001.
Supported in part by National Cancer Institute grants CA77859 (W.S.D.) and CA82533 (W.S.D.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: William S. Dalton, H. Lee Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612; e-mail: dalton{at}moffitt.usf.edu.
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© 2001 by The American Society of Hematology.
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  K. H. Shain, D. N. Yarde, M. B. Meads, M. Huang, R. Jove, L. A. Hazlehurst, and W. S. Dalton {beta}1 Integrin Adhesion Enhances IL-6-Mediated STAT3 Signaling in Myeloma Cells: Implications for Microenvironment Influence on Tumor Survival and Proliferation Cancer Res., February 1, 2009; 69(3): 1009 - 1015. [Abstract] [Full Text] [PDF]
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 P. S. Becker, K. J. Kopecky, A. N. Wilks, S. Chien, J. M. Harlan, C. L. Willman, S. H. Petersdorf, D. L. Stirewalt, T. Papayannopoulou, and F. R. Appelbaum Very late antigen-4 function of myeloblasts correlates with improved overall survival for patients with acute myeloid leukemia Blood, January 22, 2009; 113(4): 866 - 874. [Abstract] [Full Text] [PDF]
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 M. B. Meads, L. A. Hazlehurst, and W. S. Dalton The Bone Marrow Microenvironment as a Tumor Sanctuary and Contributor to Drug Resistance Clin. Cancer Res., May 1, 2008; 14(9): 2519 - 2526. [Abstract] [Full Text] [PDF]
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 L. E. Perez, N. Parquet, K. Shain, R. Nimmanapalli, M. Alsina, C. Anasetti, and W. Dalton Bone Marrow Stroma Confers Resistance to Apo2 Ligand/TRAIL in Multiple Myeloma in Part by Regulating c-FLIP J. Immunol., February 1, 2008; 180(3): 1545 - 1555. [Abstract] [Full Text] [PDF]
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 M. Kobune, H. Chiba, J. Kato, K. Kato, K. Nakamura, Y. Kawano, K. Takada, R. Takimoto, T. Takayama, H. Hamada, et al. Wnt3/RhoA/ROCK signaling pathway is involved in adhesion-mediated drug resistance of multiple myeloma in an autocrine mechanism Mol. Cancer Ther., June 1, 2007; 6(6): 1774 - 1784. [Abstract] [Full Text] [PDF]
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R. Yoshimi, S. Yamaji, A. Suzuki, W. Mishima, M. Okamura, T. Obana, C. Matsuda, Y. Miwa, S. Ohno, and Y. Ishigatsubo The {gamma}-Parvin-Integrin-Linked Kinase Complex Is Critically Involved in Leukocyte-Substrate Interaction J. Immunol., March 15, 2006; 176(6): 3611 - 3624. [Abstract] [Full Text] [PDF]
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L. A. Hazlehurst, R. F. Argilagos, M. Emmons, D. Boulware, C. A. Beam, D. M. Sullivan, and W. S. Dalton Cell Adhesion to Fibronectin (CAM-DR) Influences Acquired Mitoxantrone Resistance in U937 Cells Cancer Res., February 15, 2006; 66(4): 2338 - 2345. [Abstract] [Full Text] [PDF]
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 J. L. Rose, H. Huang, S. F. Wray, and D. G. Hoyt Integrin Engagement Increases Histone H3 Acetylation and Reduces Histone H1 Association with DNA in Murine Lung Endothelial Cells Mol. Pharmacol., August 1, 2005; 68(2): 439 - 446. [Abstract] [Full Text] [PDF]
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 J. G. Turner, R. Engel, J. A. Derderian, R. Jove, and D. M. Sullivan Human topoisomerase II{alpha} nuclear export is mediated by two CRM-1-dependent nuclear export signals J. Cell Sci., June 15, 2004; 117(14): 3061 - 3071. [Abstract] [Full Text] [PDF]
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L. A. Hazlehurst, S. A. Enkemann, C. A. Beam, R. F. Argilagos, J. Painter, K. H. Shain, S. Saporta, D. Boulware, L. Moscinski, M. Alsina, et al. Genotypic and Phenotypic Comparisons of de Novo and Acquired Melphalan Resistance in an Isogenic Multiple Myeloma Cell Line Model Cancer Res., November 15, 2003; 63(22): 7900 - 7906. [Abstract] [Full Text] [PDF]
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N. Mitsiades, C. S. Mitsiades, P. G. Richardson, V. Poulaki, Y.-T. Tai, D. Chauhan, G. Fanourakis, X. Gu, C. Bailey, M. Joseph, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications Blood, March 15, 2003; 101(6): 2377 - 2380. [Abstract] [Full Text] [PDF]
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K. H. Shain, T. H. Landowski, and W. S. Dalton Adhesion-Mediated Intracellular Redistribution of c-Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Like Inhibitory Protein-Long Confers Resistance to CD95-Induced Apoptosis in Hematopoietic Cancer Cell Lines J. Immunol., March 1, 2002; 168(5): 2544 - 2553. [Abstract] [Full Text] [PDF]
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M. M. Oshiro, T. H. Landowski, R. Catlett-Falcone, L. A. Hazlehurst, M. Huang, R. Jove, and W. S. Dalton Inhibition of JAK Kinase Activity Enhances Fas-mediated Apoptosis but Reduces Cytotoxic Activity of Topoisomerase II Inhibitors in U266 Myeloma Cells Clin. Cancer Res., December 1, 2001; 7(12): 4262 - 4271. [Abstract] [Full Text] [PDF]
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K. H. Shain and W. S. Dalton Cell Adhesion Is a Key Determinant in de Novo Multidrug Resistance (MDR): New Targets for the Prevention of Acquired MDR Mol. Cancer Ther., November 1, 2001; 1(1): 69 - 78. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
1 integrin-mediated adhesion correlates with drug
resistance in U937 cells
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Abstract
Introduction
