Ligand Bound β1 Integrins Inhibit Procaspase-8 for Mediating Cell Adhesion-Mediated Drug and Radiation Resistance in Human Leukemia Cells

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: Upon exposure to X-rays, Ara-C or FasL, suspension and adhesion (fibronectin (FN), laminin, collagen-1; 5–100 µg/cm(2) 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. CONCLUSIONS/SIGNIFICANCE: 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

Overexpression of β1 integrins mediates antiapoptotic effects in irradiated HL60 leukemia cells.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000269#s4" target="_blank">Materials and Methods</a>. Each protein fraction separated by Western blotting contains the protein amount from 2×10⁵ cells. Histon H3 was detected for the nuclear fraction and a lactate dehydrogenase (LDH) assay was performed on the cytoplasmic fraction. Numbers shown indicate the absorbance of the cytoplasmic protein fraction monitored at 490 nm and 690 nm using a spectral-photometer. (c) Level of cell surface expression of transfected β1 integrin in HL60 cells. β1 integrins were stained with FITC-conjugated anti-β1-integrin antibodies and analyzed by flow cytometry. As control, a FITC-conjugated, isotype-matched non-specific IgG (IgG control) was used. (d) Induction of apoptosis in serum grown or serum-free HL60β1 and HL60VC suspension (susp) or FN (5 µg/cm²) cell cultures was examined at 48 h after 2 or 10 Gy (mean±s.d.; n = 3). Student´s <i>t</i> test compared FN+serum, FN-serum or susp-serum versus susp+serum cultures. *<i>P</i><0.01</p

Integrin-mediated cellular resistance to X-rays and Ara-C depends on matrix protein concentrations.

<p>(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. *<i>P</i><0.01. (b) and (c) Cell viability was determined by MTT assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000269#s4" target="_blank">Materials and Methods</a>). Cells (3×10⁴) were seeded onto FN, LN or COL1 in triplicate and grown under similar conditions as described for (a). Experiments were repeated three times and results show mean±s.d.. Statistical analysis compared HL60β1 versus HL60VC cells. *<i>P</i><0.01. (d) Adhesion of HL60 transfectants to FN was evaluated in the presence or absence of stimulatory (TS2/16; 1 µg/ml) or inhibitory (13; 1 µg/ml) anti-β1 integrin mAbs or peptides (GRGDS; 500 µg/ml) under serum-free conditions (controls: non-specific anti rat IgG1 or GRADSP employed at equivalent concentrations). Columns represent mean±s.d. of the absorbance at 630 nm representing cell adhesion (n = 3). <i>P</i>-values were calculated by comparison of mAbs or peptide versus controls. *<i>P</i><0.01. (e) Radiation-induced apoptosis was determined in cells grown in suspension or on FN in the presence of TS2/16 or mAb13 (1 µg/ml; anti rat IgG1 as control) or GRGDS peptide (500 µg/ml; GRADSP as control) under serum-free conditions. Results represent mean±s.d. (n = 3) and statistics compared mAb or peptide versus controls. *<i>P</i><0.01.</p

Adhesion to matrix proteins significantly decreases induction of apoptosis in human HL60 acute promyelocytic leukemia cells after irradiation or Ara-C.

<p>(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. *<i>P</i><0.01.</p

siRNA-mediated knockdown of β1 integrin sensitizes parental, FADD-N and Casp-8N cells to radiation-induced apoptosis.

<p>(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. *<i>P</i><0.01.</p

Adhesion to FN reduces radiation-induced cleavage of procaspase-9, -3, -8 and PARP in a concentration dependent manner.

<p>(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. *<i>P</i><0.01.</p

Procaspase-8 deficiency greatly decreases radiation-induced apoptosis in FN attached cells.

<p>(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. *<i>P</i><0.01.</p

Upon adhesion, β1 integrin-mediated antiapoptotic signaling involves procaspase-8 and Akt.

<p>(a) Coprecipitation was performed to detect interactions between β1 integrin and procaspase-8, FADD or Akt. Cells were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000269#s4" target="_blank">Materials and Methods</a> and immunoprecipitation (reverse immunoprecipitation used anti-caspase-8 mAb) was carried out at 4 h after irradiation using non-specific IgG or anti-β1 integrin antibodies. (b) To analyze the impact of procaspase-8, -3 or Akt on the induction of apoptosis following FasL, HL60VC cells were held in suspension or plated onto FN and, where indicated, incubated with mAb TS2/16 or 13 (anti rat IgG1 as control) for 1 h. After 30 min, cells were also exposed to 20 µM of inhibitors for caspase-8 (IETD-fmk), -3 (DEVD-fmk), 10 µM Ly294002 (PI3K) or 0.25 µl/ml DMSO. After additional 30 min, treatment with 300 ng/ml FasL was accomplished and cells were isolated, stained with DAPI and counted for apoptotic morphology at 48 h thereafter (mean±s.d.; n = 3). Statistics were calculated by comparing inhibitor-treated cells versus DMSO or IgG. *<i>P</i><0.01. (c). In parallel, total cell extracts were isolated, subjected to Western blotting and pro and cleaved forms of caspase-8 and -3 and Akt and Akt-S473 were detected using the appropriate antibodies. β-actin was the loading control.</p