Conceived and designed the experiments: NC. Performed the experiments: NC DE AF FH. Analyzed the data: NC DE AF FH. Contributed reagents/materials/analysis tools: HS CB. Wrote the paper: HS NC CB.
The authors have declared that no competing interests exist.
Chemo- and radiotherapeutic responses of leukemia cells are modified by integrin-mediated adhesion to extracellular matrix. To further characterize the molecular mechanisms by which β1 integrins confer radiation and chemoresistance, HL60 human acute promyelocytic leukemia cells stably transfected with β1 integrin and A3 Jurkat T-lymphoma cells deficient for Fas-associated death domain protein or procaspase-8 were examined.
Upon exposure to X-rays, Ara-C or FasL, suspension and adhesion (fibronectin (FN), laminin, collagen-1; 5–100 µg/cm2 coating concentration) cultures were processed for measurement of apoptosis, mitochondrial transmembrane potential (MTP), caspase activation, and protein analysis. Overexpression of β1 integrins enhanced the cellular sensitivity to X-rays and Ara-C, which was counteracted by increasing concentrations of matrix proteins in association with reduced caspase-3 and -8 activation and MTP breakdown. Usage of stimulatory or inhibitory anti β1 integrin antibodies, pharmacological caspase or phosphatidylinositol-3 kinase (PI3K) inhibitors, coprecipitation experiments and siRNA-mediated β1 integrin silencing provided further data showing an interaction between FN-ligated β1 integrin and PI3K/Akt for inhibiting procaspase-8 cleavage.
The presented data suggest that the ligand status of β1 integrins is critical for their antiapoptotic effect in leukemia cells treated with Ara-C, FasL or ionizing radiation. The antiapoptotic actions involve formation of a β1 integrin/Akt complex, which signals to prevent procaspase-8-mediated induction of apoptosis in a PI3K-dependent manner. Antagonizing agents targeting β1 integrin and PI3K/Akt signaling in conjunction with conventional therapies might effectively reduce radiation- and drug-resistant tumor populations and treatment failure in hematological malignancies.
Integrin-mediated interactions of cells with extracellular matrix (ECM) are well known to confer resistance to clinically administered chemotherapeutic drugs or ionizing radiation
Twenty-four different α/β heterodimeric transmembrane integrin receptors are formed by 18 α and 8 β integrin subunits, which control survival, apoptosis, proliferation and differentiation among other functions in cooperation with receptor-mediated signaling from soluble growth factors or cytokines
Concerning the role of β1 integrins in adherent growing tumor and normal cells, we uncovered a signaling pathway different from the apoptosis cascades. A PI3K-dependent signaling cascade from β1 integrin to the p130Cas/Paxillin/c-Jun N2-terminal kinase complex has demonstrated to confer an advantage of clonogenic cell survival in genotoxically stressed normal fibroblasts and cells from solid tumors
In addition to anoikis, there is a large number of different apoptosis-inducing stimuli such as ionizing radiation or cytotoxic drugs. Radiation-induced genotoxic injury mainly triggers the mitochondrial cascade involving release of cytochrome c, dATP, Apaf-1 and procaspase-9 upon Bax translocation to the mitochondrial membrane that, subsequently, results in breakdown of the mitochondrial transmembrane potential (ΔΨm) and autoproteolytic cleavage of caspases
In view of the role that cell adhesion-mediated drug and radiation resistance may play in treatment failure and reduced tumor control, it becomes necessary to uncover the integrin-specific molecular mechanisms responsible for evading apoptosis. We therefore examined FasL-, radiation- and Ara-C-induced apoptosis in suspension or adhesion cultures of HL60 acute promyelocytic leukemia and Jurkat T-lymphoma cells with emphasis on integrin β1, procaspase-8 and Akt. Overexpression of the integrin β1 subunit in HL60 cells was used as a model to identify critical signaling pathways participating in the antiapoptotic action of this integrin upon cell adhesion to β1 integrin ligands such as fibronectin and collagen-1. Evidence is provided showing that a) elevated cell surface expression levels of β1 integrins inevitably require elevated amounts of ligands to act in an antiapoptotic manner, and, b) a complex formation of β1 integrin with Akt prevents procaspase-8-mediated apoptosis PI3K-dependently. These data describe a novel mechanism how the integrin β1 facilitates resistance to apoptosis induced by FasL, Ara-C and ionizing radiation, which have different modes of action.
To assess the impact of fibronectin (FN), laminin (LN) or collagen-1 (COL1) adhesion on short- and long-term survival, HL60 cells were grown in suspension or on BSA, FN, LN or COL1 prior to irradiation or Ara-C. Upon treatment, HL60 adhesion cultures on FN, LN or COL1 showed significant (
(a) At 48 h after treatment in suspension (Susp) or on BSA or FN (5 µg/cm2), cells were harvested and the number of apoptotic cells was determined by DAPI staining and counting of cells with typically apoptotic nuclear morphology. (b) Apoptosis was also determined in irradiated (10 Gy) or Ara-C (5 µM) treated HL60 cells grown on 5 µg/cm2 laminin (LN) or collagen-1 (COL1) after 48 h. (c) Limiting dilution analysis was performed to measure long-time survival. The number of positive wells (i.e. viable and proliferating cells) was used for calculation of survival rates after ionizing radiation (2, 4 or 6 Gy) or a 48-h Ara-C treatment (5 nM or 5 µM) relative to untreated controls (0 Gy or co). Results represent mean±s.d. of three independent experiments. Statistics were calculated by comparing adhesion cultures to matrix proteins versus BSA and/or suspension cultures. *
We next assessed the role of β1 integrin by stable overexpression in HL60 cells leading to an elevation in total as well as in cell surface expression of this integrin subunit as determined by Western blotting on total protein extracts (
(a) HL60 cells were stably transfected with full-length β1 integrin (HL60β1) or empty vector (HL60VC) as indicated by Western blot analysis. β-actin served as loading control. (b) Fractionation of membrane (m), cytoplasmic (c) and nuclear (n) proteins was carried out to analyze the distribution of β1 integrins in the transfected and control cells. Cells were lysed in different buffers and centrifuged according to
To clarify the adverse effect of β1 integrin-related enhancement of radiation-induced apoptosis, HL60β1 transfectants were cultured on increasing FN concentrations under serum-free conditions (
(a) After growth in suspension or FN adhesion in serum-free medium for 1 h, cells were exposed to 10 Gy X-rays or 5 µM Ara-C and apoptosis was measured 48 h thereafter (mean±s.d.; n = 3). Statistical analysis compared FN versus suspension cultures. *
To evaluate β1 integrin-dependent regulation of caspase and PARP cleavage after radiation or Ara-C, cells were analyzed on increasing FN concentrations or in suspension. At 8 h after treatment, increasing FN concentrations incrementally reduced cleavage of procaspase-9, -3 and -8 and PARP in adherent, 10-Gy irradiated HL60β1 cells relative to suspension (
(a) Following a 1-h growth on either increasing FN concentrations or in suspension, HL60β1 cells were irradiated with 10 Gy. Cells were harvested 8 h later and total proteins were extracted. After SDS-PAGE and Western blotting, selected proteins were detected using specific antibodies. β-actin served as loading control. (b) FN adhesion maintains the ΔΨm. TMRE staining of 10-Gy irradiated or Ara-C-treated (5 µM) HL60VC and HL60β1 cells was analyzed by FACS to determine the amount of ΔΨm low (mean±s.d.) representing the apoptosis-related breakdown of this potential relative to non-irradiated or non-Ara-C-treated controls ( = 0%). (c) Activation of caspases was determined by FACS analysis in FITC-VAD-fmk-stained cells under identical conditions. Results (mean±s.d. of three independent experiments) are plotted as arbitrary units (a.u.) showing the fold increase after normalization to suspension conditions. Statistics were calculated by comparison of increasing FN concentrations versus suspension. *
Coprecipitation experiments were performed showing a similar amount of precipitated β1 integrin or FADD under suspension and FN adhesion (
(a) Coprecipitation was performed to detect interactions between β1 integrin and procaspase-8, FADD or Akt. Cells were prepared as described in
Similar to suspension conditions (
To examine whether the β1 integrin-related antiapoptotic signals are channeled via procaspase-8 and Akt, Jurkat A3 cells deficient for procaspase-8 (Casp-8N) or FADD (FADD-N) were employed after inspection of protein expression (
(a) Expression of procaspase-8, FADD and β-actin was analyzed by Western blotting. Procaspase-8 negative (Casp-8N), FADD negative (FADD-N) and Jurkat A3 cells were irradiated with 10 Gy in suspension or under adhesion to 100 µg/cm2 FN. (b) Casp-8N, FADD-N and Jurkat A3 cells were exposed to mAb TS2/16 or mAb13 (1 µg/ml; anti rat IgG1 as control) for 1 h or 20 µM caspase-8 (IETD-fmk), caspase-3 (DEVD-fmk), pan-caspase inhibitor (zVAD-fmk) or 10 µM Ly294002 for 30 min when adhered to 100 µg/cm2 FN. Subsequently, cells were treated with 10 Gy or 300 ng/ml FasL. After 48 h, the number of apoptotic cells was determined by DAPI staining and counting. Columns represent mean±s.d. (n = 3). Statistical analysis was performed by comparing treatment conditions versus controls. *
For characterization of β1 integrin/procaspase-8/Akt interactions, we next performed siRNA-mediated knockdown of β1 integrin prior to X-ray or FasL exposure. Two different siRNAs (β1.1, β1.2) mediated β1 integrin silencing (β1.1: 90–98% repression; β1.2: 80–95% repression) relative to non-specific Duplex XII (
(a) Jurkat cell lines were transfected with two different β1 integrin (β1.1, β1.2) siRNAs or a non-specific Duplex XII (DXII) siRNA. Expression of β1 integrin was inspected by immunoblotting. (b) Following β1 integrin knockdown, 10 Gy or 300 ng/ml FasL were applied to the cells grown on 100 µg/cm2 FN. Apoptosis was determined 48 h later by DAPI. (c) In parallel, cell lysates were harvested for analysis of procaspase-8, -3 and Akt expression. (d) Subsequent to administration of 20 µM caspase-8 (IETD-fmk) or caspase-3 (DEVD-fmk), 10 µM Ly294002 or 0.25 µl/ml DMSO for 30 min, caspase-8 and -3 activity was measured at 4 h after 10 Gy. Statistics were calculated by comparing the level of apoptosis in β1 integrin knockdown cells versus DXII. *
In contrast to Duplex XII controls, caspase-8 activity in FN adherent and irradiated A3 or FADD-N Jurkat cells was significantly (
Chemo- and radiotherapeutic responses of leukemia cells are essentially modified by integrin-mediated adhesion to extracellular matrix
Unexpectedly in its extent, increases in β1 integrin total and cell surface expression inevitably required increased availability of a ligand, here fibronectin, laminin or collagen-1, for sufficient antiapoptotic action after different types of cell stress such as ionizing radiation, FasL or Ara-C. To note, serum depletion also reduced the rate of radiation-induced apoptosis. It can be hypothesized that specific growth factors are critical for the accurate execution of proapoptotic pathways. Extensive experiments have already commenced in our laboratory to elucidate this observation in more depth. By parallel modulation of both the intrinsic and extrinsic apoptotic pathway, the functional duality of the integrin β1 subunit in prosurvival processes is exceptionally displayed in our study.
Owing to recent findings on procaspase-8 in integrin-mediated death
To evaluate these effects in Jurkat cells deficient for the critical molecules of the death receptor cascade, i.e. procaspase-8 and FADD, procaspase-8 and FADD deficient cells were tested. FADD-negative cells reacted, in general, similar to A3 Jurkat control cells under adhesion conditions. Blocking caspase activation by pharmacological inhibitors reduced radiation- and FasL-induced apoptosis in contrast to Ly294002. PI3K deactivation resulted in elevated levels of apoptosis under all tested treatment regimes. As this indicates that the effect is procaspase-8- but not FADD-dependent, procaspase-8 deficient Jurkat cells showed less apoptosis throughout the diverse treatment and growth conditions tested but retained some of their susceptibility to β1 integrin modification by anti-β1 mAbs. Despite data that describe FADD recruitment to FasR in the absence of FasL for activating procaspase-8 after anticancer drugs or UV-irradiation
Accomplishing knockdown of β1 integrin by siRNA increased the sensitivity of Jurkat A3, FADD-N, and Casp-8N cells particularly to X-rays and to a lesser extent to FasL. In addition to pronounced augmentation in caspase-8 and -3 activity, elevated cleavage of procaspase-8 and -3 was associated in all cases with an attenuated phosphorylation of Akt at S473.
In summary, our data demonstrate, for the first time as to our knowledge, a regulatory interaction between β1 integrin, Akt and procaspase-8 selectively assembled after integrin-mediated adhesion of leukemia cells to FN. Due to its critical role in interfering with apoptosis-triggering agents such as ionizing radiation, FasL and Ara-C, this complex might essentially contribute to pre-existing or acquired resistance mechanisms effectively counteracting current antitumor therapies. Both, agents targeting β1 integrin signaling and agents targeting the PI3K/Akt pathway might represent potent novel adjuvant therapeutic options. Application of such agents in conjunction with conventional therapies might effectively reduce drug-resistant tumor populations and treatment failure in hematological malignancies.
All reagents were purchased from commercial sources: 4′,6 Diamidino-2-phenylindole (DAPI; Serva, Heidelberg, Germany), FITC-VAD-fmk (Promega, Mannheim, Germany), TMRE (tetramethylrhodamine, ethyl ester, perchlorate), MitoTracker® Red CMXRos (Molecular Probes, Leiden, Netherlands), zVAD-fmk, Ly294002, protein-G-agarose beads, diaminobenzidine (Sigma, Taufkirchen, Germany), DEVD-fmk, IETD-fmk (Chemicon, Hampshire, UK), GRGDS (H-Gly-Arg-Gly-Asp-Ser-OH) and GRADSP (H-Gly-Arg-Ala-Asp-Ser-Pro-OH) peptides, G418 (Calbiochem, Bad Soden, Germany), Vectashield® medium (Alexis, Grünberg, Germany), nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany), ECL (Amersham, Freiburg, Germany). Antibodies used are: anti-β1 integrin (TS2/16; Perbio, Bonn, Germany), anti-rat IgG1, anti-mouse IgG1 (Upstate, Hamburg, Germany), anti-caspase-3 cleaved, anti-caspase-3, anti-caspase-9 cleaved, anti-caspase-9, anti-caspase-8 cleaved, anti-caspase-8, anti-PARP cleaved, anti-PARP, anti-FADD, anti-Akt-S473, anti-Akt (Cell Signaling, Frankfurt a.M., Germany), anti-β-actin (Sigma, Taufkirchen, Germany), anti-β1 integrin (BD, Heidelberg, Germany); HRP-conjugated goat anti-rabbit and anti-mouse antibodies (Santa Cruz, Heidelberg, Germany). Anti-β1 integrin (13) was a generous gift from K.M. Yamada (Bethesda, MA, USA). FITC conjugated anti-β1-integrin IgG and FITC-conjugated non-specific anti-IgG antibodies were from Dako (Hamburg, Germany). Anti-Histon H3 was from Acris (Hiddenreich, Germany). Human promyelocytic HL60 leukemia and Jurkat A3 T-lymphoma cells were purchased from ATCC (Bethesda, MD, USA). Caspase-8- and FADD-deficient Jurkat A3 cells were a kind gift from P. Juo and J. Blenis (Boston, MA, USA). RPMI-1040 GlutaMAX 1TM supplemented with 1% non-essential amino acids (GIBCO, Karlsruhe, Germany) and 10% FCS (PAA, Linz, Austria) was applied to culture the cells routinely at 37°C-5% CO2, pH 7.4. Serum starvation of cells was performed using RPMI-1040/1% non-essential amino acids without FCS. For all experiments, asynchronous growing cell cultures were used.
The full-length of human β1 integrin cDNA was generated by PCR and cloned into the pcDNA3 expression vector using EcoR1 sites (Invitrogen, Karlsruhe, Germany). Subsequent to electroporation
Cells were induced to undergo apoptosis using ionizing radiation, Ara-C or FasL. Cells were grown in suspension (polystyrene, BSA (bovine serum albumin)) or on FN (BD, Heidelberg, Germany) plus/minus serum for 1 h, irradiated or left unirradiated, treated with Ara-C (0, 5 nM, 5µM) or FasL (300 ng/ml; Merck, Germany), prepared by cytospin, washed with 0.9% NaCl and permeabilized using 4% paraformaldehyde for morphological evaluation of apoptosis by DAPI staining as published
In average, one cell was plated in every non-coated or FN- or BSA-precoated well of a microtiter plate. After 1 h, irradiation (0, 2, 4, 6 Gy) or Ara-C (48 h; 0, 5 nM, 5 µM) was delivered and cells were allowed to grow for 8 days according to Grenman et al.
For fractionation of membrane, cytoplasmic and nuclear proteins, cells were lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM EDTA, protease inhibitor cocktail complete® (Roche, Mannheim, Germany)) and sonicated (2×1 sec, level 4, 60%) on ice and cytoplasmic proteins were separated from nuclear and membrane proteins by centrifugation (100,000×g, 15 min, 4°C). Then, the pellet was resuspended in Triton X-100 buffer (1% Triton X-100, 10 mM MgCl2, 0.2 mM Na3VO4, protease inhibitor cocktail complete®) to separate membrane proteins from nuclear proteins by centrifugation (23,000×g, 5 min, 4°C). After removal of the supernatant containing the membrane faction, the pellet was resuspended in loading buffer (50 mM Tris-Base (pH 6.8), 2 ml Glycerol, 10% SDS, 0.5 ml β-mercaptoethanol, 1 mg bromphenol blue). Each protein fraction separated by Western blotting contained the protein amount from 2×105 cells. To verify accurate protein fractionation, Histon H3 was detected in the nuclear fraction and a lactate dehydrogenase (LDH) assay (Roche, Mannheim, Germany) was performed on the cytoplasmic fraction. Samples were prepared according to the manufacturer's instructions. Absorbance at 490 nm and 690 nm was monitored using a spectral-photometer (Spectra max® 190).
The expression level of transfected β1 integrins was measured by FACS analysis as published
Cell adhesion to FN was studied according to a previously published method
At 24 h after treatment, cells were prepared for measurement of the ΔΨm using 25 nM TMRE and flow cytometry following the manufacturer's instructions as published
Analysis of activated caspases was performed as previously described using FITC-VAD-fmk and flow cytometry
After 10-Gy X-rays or 300 ng/ml FasL, suspension and FN adhesion cultures were harvested and lysed on ice using 50 mM Tris-HCL (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail complete®, 5 mM sodium vanadate and 5 mM sodium fluoride. Amounts of total protein extracts were determined using BCA assay (Interchim, Montlucon Cedex, France) and samples were stored at −134°C until use. Western blotting was performed as described previously
Caspase-3 or -8 activities were measured after 10-Gy X-rays plus/minus DEVD-fmk or Ly294002 (control: DMSO) in triplicates using a commercially available ApoAlert assay kit (BD-CloneTech, Heidelberg, Germany) or Caspase-8 Colorimetric Activity Assay Kit (Chemicon, Ochsenhausen, Germany) according to the manufacturer's instructions. Experiments were repeated three times.
Cells were grown in suspension or on 100 µg/cm2 FN in serum-free medium 1 h before 10-Gy radiation. Then, cells were treated for 15 min with 1% formaldehyde to crosslink proteins, a reaction terminated with 100 mM glycine. Following cell lysis, β1 integrin was immunoprecipitated with 2 µg of the specific antibody overnight at 4°C from 250 µg total protein extracts. Subsequently, protein-G-agarose beads were allowed to incubate for 3 h, followed by washing and preparation for SDS-PAGE. β1 integrin and coprecipitated procaspase-8, FADD or Akt were detected by Western blotting. Non-specific mouse-IgG was used as control.
The target sequences that effectively mediate silencing of β1 integrin expression are 5′-AATGTAACCAACCGTAGCA-3′ (β1.1) and 5′-GCGCATATCTGGAAATTTG-3′ (β1.2) (sense sequences) as reported previously
Means±s.d. of three independent experiments were calculated with reference to untreated controls defined in a percentage scale or 1.0. To test statistical significance, Student´s
We thank B. Reincke, M. Hiber, G. Schröder and M. Kraus for excellent technical assistance. The authors are indebted to K.M. Yamada for providing mAb13 (Bethesda, MA, USA) and P. Juo and J. Blenis (Boston, MA, USA) for the caspase-8- and FADD-negative Jurkat cells.