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[Cancer Research 64, 5398-5406, August 1, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Differential Activation of the Phosphatidylinositol 3'-Kinase/Akt Survival Pathway by Ionizing Radiation in Tumor and Primary Endothelial Cells

Daniel Zingg1, Oliver Riesterer1, Doriano Fabbro2, Christoph Glanzmann1, Stephan Bodis1 and Martin Pruschy1

1 Department of Radiation Oncology, University Hospital Zurich, Zurich, and 2 Novartis Pharma, Inc., Department of Oncology Research, Basel, Switzerland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ionizing radiation induces an intracellular stress response via activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt survival pathway. In tumor cells, the PI3K/Akt pathway is induced through activation of members of ErbB receptor tyrosine kinases. Here, we investigated the receptor dependence of radiation-induced PI3K/Akt activation in tumor cells and in endothelial cells. The integrity of both the ErbB and the vascular endothelial growth factor (VEGF) ligand-activated PI3K/Akt pathway in endothelial cells was demonstrated using specific ErbB and VEGF receptor tyrosine kinase inhibitors. Irradiation of endothelial cells resulted in protein kinase B (PKB)/Akt activation in a similar time course as observed in response to VEGF. More importantly, radiation-induced PKB/Akt phosphorylation in endothelial cells was strongly down-regulated by the VEGF receptor tyrosine kinase inhibitor, whereas the ErbB receptor tyrosine kinase inhibitor did not affect PKB/Akt stimulation in response to irradiation. An opposite receptor dependence for radiation-induced PKB/Akt phosphorylation was observed in ErbB receptor-overexpressing A431 tumor cells. Furthermore, direct VEGF receptor phosphorylation was detected after irradiation in endothelial cells in absence of VEGF, which was almost completely inhibited after irradiation in presence of the VEGF receptor tyrosine kinase inhibitor. These data demonstrate that ionizing radiation induces VEGF ligand-independent but VEGF receptor-dependent PKB/Akt activation in endothelial cells and that PI3K/Akt pathway activation by radiation occurs in a differential cell type and receptor-dependent pattern.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ionizing radiation (IR) induces a complex network of signaling processes at cellular sites distant from and independent of DNA damage and many of these processes unveil new therapeutic interventions to combine IR with pharmacological anti-signaling agents. IR-induced activation of receptor tyrosine kinases (RTKs) is a highly investigated area to understand the cellular stress response to IR (1, 2, 3, 4) . Similar to growth factor stimulation, irradiation results in defined phosphorylation of Tyr residues in the cytoplasmic domain of RTKs, which serve as docking sites for signaling entities of additional downstream signal transduction pathways such as the ras/mitogen-activated protein kinase, phosphatidylinositol 3'-kinase (PI3K), and signal transducers and activators of transcription 3 (STAT-3) pathways, which are involved in enhanced proliferation and regulate an intrinsic survival threshold. These signaling activities might contribute to radiation-induced accelerated tumor cell repopulation and enhanced radioresistance (5 , 6) . These IR-induced processes represent an additional rationale that RTKs are promising targets for combined treatment modalities using IR in combination with RTK inhibitors. A variety of promising approaches were developed to abrogate the signaling processes generated from mutated and overexpressed members of the epidermal growth factor (EGF) or ErbB family of type I RTKs such as EGF receptor (EGFR) (ErbB1/HER1) and HER2 (ErbB2/HER2). These include strategies that target the extracellular RTK domains, e.g., with antireceptor antibodies or that inhibit the intracellular tyrosine kinase activity with low molecular weight inhibitors (7, 8, 9, 10) . Both approaches resulted in compounds that have been tested in preclinical studies and that have entered clinical trials during recent years, alone, and in combination with chemotherapy and radiotherapy (11 , 12) .

The PI3K/Akt signaling pathway is activated through growth factor- and radiation-induced ErbB receptor stimulation in tumor cells and is involved in the cellular response to stress stimuli and apoptosis regulation (13, 14, 15, 16, 17) . PI3K can directly be recruited to the specific phosphorylated docking sites of the ErbB3 isoform of the EGFR family through the SH2-domain of its p85 regulatory subunit (18, 19, 20) . Because of the lack of an intact kinase domain ErbB3 requires heterodimerization with other ErbB isoforms such as ErbB1 (EGFR) or ErbB2, which by themselves lack specific consensus sites for PI3K binding. However, PI3K can also be stimulated in an ErbB3 independent way through the interaction of RTK-activated ras with PI3K (21) .

One of the downstream targets of PI3K is the serine-threonine protein kinase PKB/Akt also referred to as protein kinase B (PKB or RAC). Activation of PKB/Akt involves the binding of PI3K-phosphorylated phosphoinositides to the PKB/Akt-pleckstrin domain and translocation to the plasma membrane, where PKB/Akt is phosphorylated by the phosphatidylinositide (PI)-dependent kinase PDK1 and an unidentified kinase referred as PDK2. Phosphorylation of the two phosphorylation sites Thr308 by PDK1 and Ser473 is required for its activation (22 , 23) . Several downstream targets of PKB/Akt have been identified (e.g., the bcl-2 family member Bad, caspase-9, mdm2, the transcription factor forkhead, and glycogen synthase kinase 3), thus conferring to PKB/Akt an important role in the regulation of downstream growth-promoting and cell survival signaling pathways (24, 25, 26, 27, 28) .

Targeting the vascular system of tumors has been regarded as a highly promising anticancer strategy not only because it is directed against the delivery of nutrients, growth factors, and oxygen but also because it prevents tumor progression and the formation of metastasis (29 , 30) . Furthermore, targeting the vascular system is an interesting approach because primary endothelial cells of the tumor angiogenic system do not undergo genetic mutations. In particular, specific tyrosine kinase inhibitors of the vascular endothelial growth factor receptor (VEGFR) have been developed as inhibitors of angiogenesis (31, 32, 33, 34, 35) . This is mainly due to the important role of the VEGF as a proangiogenic factor in pathological situations, neovascularization and enhanced vascular permeability, and the advanced mechanistic understanding of the corresponding VEGFRs. The VEGFRs Flt-1 (Fms-like tyrosine kinase, VEGFR-1) and KDR (kinase insert domain-containing receptor, VEGFR-2) are predominantly located on endothelial cells (36) . Both receptors have seven immunoglobulin-like domains in their extracellular region, a single transmembrane-spanning domain, and an intracellular split tyrosine kinase domain and belong to the same family of receptors as EGFR, platelet-derived growth factor receptor, c-Kit, c-Fms, Flt-3, and Flt-4. Flt-1 binds VEGF-A and VEGF-B and the related placenta growth factor, whereas KDR binds VEGF-A, VEGF-C, and VEGF-D (37 , 38) . KDR is strongly autophosphorylated upon VEGF stimulation, and VEGF induces signaling processes such as the PI3K/Akt pathway through direct recruitment of downstream targets to the phosphorylated consensus sites of the VEGFR (39, 40, 41)

Interestingly, IR also activates PKB/Akt in endothelial cells in a PI3K-dependent way, and PI3K inhibitors enhance radiation-induced apoptosis and cytotoxicity in these cells (42) . Thus, inhibition of the radiation-activated PI3K/Akt survival pathway might also contribute to an increased antiangiogenic effect after treatment with IR in combination with inhibitors of this survival pathway. However, radiation-induced activation of the PI3K/Akt pathway could be mediated through different upstream RTKs, and current PI3K inhibitors (e.g., wortmanin, LY294002) are overly toxic for clinical use (43) . We therefore investigated radiation-induced PI3K/Akt stimulation in endothelial cells using clinically relevant RTK inhibitors and compared its activation profile to radiation-induced PKB/Akt stimulation in tumor cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures, Irradiation, and Treatment with RTK Inhibitors.
The vulvar squamous carcinoma cell line A431 was grown at 37°C and 5% CO2 atmosphere in DMEM containing 10% FCS (Life Technologies Laboratories, Karlsruhe, Germany) supplemented with penicillin and streptomycin. For serum starvation, A431 cells were washed with PBS and incubated with DMEM containing 0.5% FCS for 24 h. The primary human umbilical vein endothelial cells (HUVECs; PromoCell GmbH, Heidelberg, Germany) were used between the second and the seventh passage number and cultured in endothelial cell growth medium kit (EGM; PromoCell GmbH) containing 5% FCS, 0.4% endothelial cell growth supplement/H, 0.1 ng/ml EGF, 1 ng/ml basic fibroblast growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. HUVECs were plated in collagen I coated 6 cm (Becton Dickinson Labware, Bedford, MA) or 1.5% gelatin-coated 10-cm Petri dishes. For growth factor starvation, HUVECs were washed with PBS and incubated with endothelial cell basal medium (EBM; PromoCell GmbH) supplemented with 0.1% BSA, amphotericin B, and penicillin/streptomycin, for 12 h. The human dermal microvascular endothelial cells (PromoCell GmbH) were used between the second and the seventh passage number and cultured in endothelial cell growth medium kit (EGM MV; PromoCell GmbH) containing 5% FCS, 0.4% endothelial cell growth supplement/H, 10 ng/ml EGF, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, and 50 ng/ml amphotericin B. Growth factor starvation for human dermal microvascular endothelial cells was performed as described for HUVECs. EGF and human VEGF165 (R&D Systems, Wiesbaden-Nordstadt, Germany) were added to the culture media for the indicated time to a final concentration of 50 and 20 ng/ml, respectively. Antihuman EGF and antihuman VEGF antibodies were purchased from R&D Systems and added to the culture media as indicated. Irradiation was carried out at room temperature using a Pantak Therapax 300kV X-ray unit at 0.7 Gy/min. Cellular pretreatment with PKI166 (100 or 500 nM; Novartis-Pharma, Basel, Switzerland) or PTK787/ZK222584 (100 nM; Novartis-Pharma) was performed for 1 and 2 h before irradiation, respectively.

Immunoblotting.
Cells were harvested at different time points by scraping off the cells in 100 µl of SDS sample buffer. Samples were stored at –80°C or directly resolved by SDS-PAGE followed by blotting onto polyvinylidene difluoride membranes. Membranes were probed with rabbit polyclonal anti-PKB/Akt, anti-PKB/Akt-phosphoSer473 (New England Biolabs, Beverly MA), anti-ErbB1-phosphoTyr1086 (Calbiochem, Darmstadt Germany) or anti-VEGFR-2-phosphoTyr951 antibodies (Cell Signaling, Beverly, MA), or mouse monoclonal anti-ß-actin (clone AC-15; Sigma, Buchs Switzerland), anti-ErbB1 (Oncogene, Boston, MA), and anti-VEGFR-2-antibodies (Calbiochem). Antibody detection was achieved by enhanced chemiluminescence-enhanced chemiluminescence (Amersham, Freiburg, Germany) using a horseradish peroxidase-conjugated second antibody, according to the manufacturer’s protocol.

Immunoprecipitation.
Cells were washed with ice-cold PBS and harvested by scraping off cells in 500 µl of buffer A [20 mM Tris-HCl (pH 8.0), 5% Glycerol (m/V), 1% NP40 (m/V), 2.7 mM KCl, 138 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 50 mM sodium EDTA, and 1 mM DTT], supplemented with protease and phosphatase inhibitors (5 mg/ml pepstatin A, 10 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mM Na3VO4, 1 mM NaF, and 0.1 mM phenylmethylsulfonyl fluoride). After incubation on ice for 30 min, the lysate was centrifugated at 15,000 x g for 10 min at 4°C. Protein concentration was determined from the supernatant by colorimetric measurement using the Bio-Rad DC protein assay kit II according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA). Supernatants (1 mg/ml protein) were incubated with 25 µg of protein A agarose-conjugated anti-phosphoTyr monoclonal antibody (4G10; Upstate Biotechnology, Lake Placid, NY) or 2 µg of anti-VEGFR-2 polyclonal antibody (Calbiochem) overnight at 4°C and with protein A-conjugated agarose beads (Upstate Biotechnology) at 4°C for 4 h. The beads were washed three times in 0.5 ml of buffer A, supplemented with all inhibitors, and immunoprecipitates were resuspended in 2x SDS sample buffer for Western blot analysis.

Immunofluorescence Labeling and Confocal Laser-Scanning Microscopy.
HUVECs were treated in 2-well collagen-coated plates (Biocoat; Becton Dickinson) as indicated above, followed by fixation with 3% paraformaldehyde for 15 min and cell permeabilization with 0.5% Triton X-100 in PBS for 2.5 min. The primary rabbit polyclonal anti-VEGFR-2 phosphoTyr996 antibody (Calbiochem) was used at a dilution of 1:50. The secondary Texas Red X-conjugated affinity-purified goat antirabbit IgG (H + L) antibody (Molecular Probes, Eugene, OR) was used at a dilution of 1:100. Both antibodies were incubated for 1 h with the cells. Cells were costained with 4',6-diamidino-2-phenylindole (10 µg/ml) for detection of nuclei. The coverslips were mounted on glass slides and embedded in Mowiol (Calbiochem). All steps were performed at room temperature. A Zeiss Axioplan fluorescence microscope (Zeiss, Jena, Germany) equipped with a confocal scanning unit MRC-600 (Bio-Rad Laboratories, Herts, United Kingdom) and an argon-krypton laser was used to acquire images, which were subsequently processed using Adobe PhotoShop, system 6.0 (San Jose, CA) and reconstructed using simple PC software.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IR Activates PKB/Akt in Human Endothelial Cell.
The PI3K/Akt survival pathway is activated in different cell lines in response to various stress factors. PKB/Akt activation is a multistep process and includes phosphorylation of PKB/Akt at the specific residue Ser473 also in response to irradiation with IR (44) . To determine PI3K/Akt pathway activation in endothelial cells, we investigated the PKB/Akt phosphorylation status in HUVECs after treatment with low doses of IR using the Ser473 site-specific anti-phospho-Akt antibody. HUVECs were serum starved for 12 h, and the PKB/Akt phosphorylation status was then analyzed by Western blotting of whole cellular lysates after irradiation with 2 Gy (Fig. 1A)Citation . A clear increase in the phosphorylation level was observed at 20, 30, 60, and 120 min after irradiation, which is in agreement with recent data reported by Edwards et al. (42) . A dose-dependent effect of PKB/Akt phosphorylation was investigated after treatment with increasing doses of IR. Interestingly, irradiation with doses as high as 10 Gy did not additionally increase the Ser473-PKB/Akt phosphorylation level (Fig. 1B)Citation .



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  		Fig. 1. Ionizing radiation induces PKB/Akt phosphorylation in human endothelial cells. A, after 12 h of serum deprivation, human umbilical vascular endothelial cells (HUVECs) were irradiated with 2 Gy radiation and analyzed at the indicated time points. The phosphorylation level of PKB/Akt was determined in whole cell extracts using an anti-PKB/Akt phosphoSer473 site-specific antibody. B, cells were irradiated with increasing doses of ionizing radiation (IR) and PKB/Akt phosphorylation level was determined at the 30-min time point as in A. Samples were reanalyzed using anti-ß-actin or anti-Akt antibodies to ensure the loading of equal amounts of protein.

 
The PI3K/Akt pathway is activated in proliferating cells via growth factor receptor stimulation, and interestingly, IR-induced PKB/Akt phosphorylation has been demonstrated in epithelial tumor cells to be mediated through activation of ErbB receptors (13) . Thus, IR might also induce PKB/Akt phosphorylation in HUVECs via upstream RTKs. To investigate the mechanism of IR-dependent PKB/Akt activation in greater detail, we analyzed at first the effect of PKB/Akt phosphorylation in endothelial cells in response to the two intrinsic ligands of the ErbB and VEGFR systems.

HUVECs were serum deprived for 12 h before treatment with the two growth factors EGF (50 ng/ml) and VEGF (20 ng/ml), respectively, followed by analysis of the Ser473-Akt phosphorylation status by Western blotting of whole cellular protein extracts. PKB/Akt phosphorylation could be observed 5 and 10 min after EGF stimulation, whereas growth factor-specific up-regulation of the PKB/Akt phosphorylation status was only observed as early as 10 min after stimulation with VEGF (Fig. 2)Citation . The delayed kinetics of PKB/Akt phosphorylation in response to VEGF mirrored PKB/Akt stimulation after irradiation. Furthermore, these results show that the PI3K/Akt pathway can be activated both via EGF or VEGF in endothelial cells.



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  		Fig. 2. Epithelial growth factor (EGF) and vascular epithelial growth factor (VEGF) activate PKB/Akt with a different kinetic in human endothelial cells. After 12 h of serum deprivation, human umbilical vascular endothelial cells (HUVECs) were stimulated with EGF (50 ng/ml; A) or with VEGF (20 ng/ml; B) for the indicated times. The phosphorylation level of PKB/Akt was determined in whole cell extracts using an anti-PKB/Akt phosphoSer473 site-specific antibody. Samples were reanalyzed with an anti-ß-actin antibody to ensure the loading of equal amounts of protein.

 
VEGF and ErbB Receptor-Mediated Activation of PKB/Akt.
Clinically relevant inhibitors of the VEGF receptor system (the phtalazine derivative PTK787/ZK222584) and the ErbB1/2 receptors (the pyrrolopyrimidine derivative PKI166) were used to dissect whether activation of specific RTKs is required for IR-induced PKB/Akt phosphorylation. On the basis of the high selectivity of these rational designed pharmacological compounds, they are suitable as small molecular probes to dissect signal transduction cascades. Both the ErbB1/2 inhibitor PKI166 and the VEGFR inhibitor PTK787/ZK222584 inhibit the kinase activity of their respective receptors in the submicromolar range, with an overlapping inhibitory effect only at higher doses levels (7) . To determine the required concentration for selective receptor inhibition, growth factor-dependent PKB/Akt activation was first tested in HUVECs and the ErbB1-overexpressing human tumor cell line A431. Both cell lines were serum starved for 12 h and stimulated with EGF (50 ng/ml) with or without pretreatment with PKI166 (500 nM) or PTK787/ZK222584 (100 nM) for 1 h. EGF-induced PKB/Akt phosphorylation was completely abrogated in A431 and HUVECs, respectively, when cells were pretreated with PKI166 (Fig. 3, A and BCitation ; 500 nM). On the other hand, cellular pretreatment with PTK787/ZK222584 (100 nM) did not affect EGF-induced PKB/Akt phosphorylation in either cell line (Fig. 3, C and D)Citation . Furthermore, PKB/Akt phosphorylation was also tested in HUVECs in response to VEGF. Although the ErbB inhibitor PKI166 did not down-regulate VEGF-dependent PKB/Akt phosphorylation, the phosphorylation status was strongly decreased on pretreatment with the VEGF receptor inhibitor PTK787/ZK222584 (Fig. 3E)Citation . These results confirm that EGF- and VEGF-induced PKB/Akt activation are mediated through their corresponding RTKs and demonstrate the growth factor receptor-selective inhibitory potential of the two RTK inhibitors to dissect the potential link between RTKs and IR-induced PI3K/Akt stimulation in tumor and endothelial cells.



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  		Fig. 3. Vascular epithelial growth factor receptor (VEGFR)- and ErbB receptor-dependent activation of PKB/Akt. Serum-starved A431 (A and C) and human umbilical vascular endothelial cells (HUVECs; B and D) were preincubated in the absence or presence of 0.5 µM PKI166 (A and B) and 100 nM PTK787/ZK222584 (C and D) followed by stimulation with epithelial growth factor (EGF; 50 ng/ml). The phosphorylation level of PKB/Akt was determined in whole cell extracts using an anti-PKB/Akt phosphoSer473 site-specific antibody. E, after 12 h of serum-deprivation, HUVECs were preincubated with 100 nM PTK787/ZK222584 or 0.5 µM PKI166 and stimulated with VEGF (20 ng/ml) for 15 min. The PKB/Akt phosphorylation level was quantified as mentioned above. In all experiments, samples were reanalyzed with an antibody against ß-actin to ensure the loading of equal amounts of protein.

 
Receptor-Dependent PKB/Akt Stimulation by IR in Tumor and Endothelial Cells.
Using these pharmacological inhibitors we tested RTK-dependent PKB/Akt phosphorylation after irradiation in endothelial cells and in the tumor cell line A431. A431 cells were serum starved, pretreated with PKI166 (500 nM) or PTK787/ZK222584 (100 nM) for 1 h and irradiated with 5 Gy of IR. The ErbBR inhibitor completely abrogated IR-induced PKB/Akt phosphorylation whereas the VEGFR inhibitor did not affect PKB/Akt phosphorylation in these cells (Fig. 4, A and B)Citation . These results are in accordance with previous data demonstrating that IR activates PKB/Akt in tumor cells via an ErbB receptor-mediated process (13 , 45 , 46) .



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  		Fig. 4. Differential receptor-dependent PKB/Akt stimulation by ionizing radiation in tumor and endothelial cells. A431 (A and B), human umbilical vascular endothelial cells (HUVECs; C and D), and HDME cells (E and F) were serum deprived for 24 or 12 h, respectively, followed by preincubation in the absence or presence of 0.5 µM PKI166 (A, C, and E) or 100 nM PTK787/ZK222584 (B, D, and F) and irradiated with 5 Gy of IR. The phosphorylation level of PKB/Akt was determined in whole cell extracts using an anti-PKB/Akt phosphoSer473 site specific antibody 30 min after irradiation. Samples were reanalyzed with an antibody against ß-actin to ensure the loading of equal amounts of protein. All experiments were repeated at least twice.

 
In parallel, PKB/Akt stimulation was investigated in two endothelial cell lines. Interestingly, PKI166 did not decrease the IR-induced PKB/Akt phosphorylation status in HUVECs, whereas pretreatment with PTK787/ZK222584 down-regulated IR-dependent PKB/Akt phosphorylation (Fig. 4, C and D)Citation . Likewise, IR-induced PKB/Akt phosphorylation in microvascular endothelial cells, which show a different proliferative activity than umbilical vein endothelial cells, was also sensitive to PTK787/ZK222584 but not to PKI166 (Fig. 4, E and F)Citation . These data strongly suggest a differential RTK-dependent stimulation of the PI3K/Akt pathway by IR in tumor and endothelial cells, respectively.

This differential effect was identified in endothelial cells, which were cultured in basal medium in absence of serum and the respective growth factors VEGF or EGF. We additionally tested IR-induced receptor-mediated PKB/Akt stimulation under enriched growth conditions (5% FCS, EGF, endothelial cell growth supplement, and hydrocortisone) in absence of serum withdrawal-based stress. The basal phosphorylation status of PKB/Akt was thereby enhanced most probably due to intact EGF/ErbB-mediated signaling present in HUVECs as already determined (Fig. 5ACitation , see also Fig. 3BCitation ). To investigate ErbB-ligand independent PKB/Akt phosphorylation under these conditions, cellular irradiation was performed after preincubation with the ErbB-RTK inhibitor PKI166 (500 nM), which reverted serum and growth factor induced PKB/Akt phosphorylation (Fig. 5ACitation , Lanes 1 and 2). Irradiation additionally enhanced PKB/Akt phosphorylation well above basal level also in presence of PKI166. On the other hand, PKB/Akt-phosphorylation did not occur when cells were pretreated with the VEGF RTK inhibitor PTK787/ZK222584 (Fig. 5ACitation , Lanes 4 and 5.) These results corroborate VEGFR-mediated PKB/Akt phosphorylation in response to irradiation also in cells that did not undergo serum withdrawal-based stress.



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  		Fig. 5. Ligand-independent receptor-mediated PKB/Akt stimulation by ionizing radiation. In A, human umbilical vascular endothelial cells (HUVECs) were cultured under serum and growth factor-enriched growth conditions and preincubated in presence or absence of PKI166 (0.5 µM, 3 h) and PTK787/ZK222584 (100 nM, 2 h) before irradiation with 5 Gy. For growth factor trapping A431 cells (B) or HUVECs (C) were pretreated with an anti-epithelial growth factor (EGF; 0.5 µg/ml) or an anti-vascular epithelial growth factor (VEGF) antibody (0.5 µg/ml), respectively, added twice (5 and 30 min) before stimulation with EGF (1 ng/ml), VEGF (10 ng/ml), or irradiation (5 Gy). In D and E, A431 cells (D) and HUVECs (E) were serum deprived for 24 or 12 h, respectively, followed by irradiation with 5 Gy. PKI166 (0.5 µM; D) or PTK787/ZK222584 (0.1 µM; E) was added to the respective culture medium 20 min after irradiation. The phosphorylation level of PKB/Akt was determined in all whole cell extracts using an anti-Akt phosphoSer473 site-specific antibody 30 min after irradiation. Samples were reanalyzed with an antibody against ß-actin to ensure the loading of equal amounts of protein. All experiments were repeated at least twice.

 
To investigate whether IR-induced PKB/Akt activation might be induced through a ligand-mediated RTK activation process A431 tumor cells and HUVECs were incubated with {alpha}-EGF-antibodies and {alpha}-VEGF-antibodies, respectively, before irradiation to trap potentially stress-released growth hormones (Fig. 5, B and C)Citation . Control experiments were performed demonstrating that cellular preincubation with these antibodies diminishes PKB/Akt phosphorylation when cells were stimulated with the respective growth factors EGF or VEGF (Fig. 5CCitation , Lanes 1–3). However, preincubation of tumor cells with {alpha}-EGF antibodies and endothelial cells with {alpha}-VEGF antibodies did not decrease IR-induced PKB/Akt stimulation in these cells (Fig. 5CCitation , Lanes 4 and 5). Although we cannot completely exclude that different growth factors, which are not trapped by these antibodies, were liberated and activated these receptors in response to IR, these results strongly support that RTK-dependent PKB/Akt stimulation in response to irradiation does occur through direct activation of the receptor by irradiation independently of a ligand-mediated effect.

We tested whether cellular treatment with the RTK inhibitors after irradiation reverses PKB/Akt phosphorylation. HUVECs and A431 tumor cells were irradiated and treated with the respective inhibitors followed by analysis of the PKB/Akt phosphorylation status. Radiation-induced PKB/Akt phosphorylation was not reversed in HUVECs when PTK787/ZK222584 was added after irradiation. Interestingly though, PKI166 treatment did abrogate IR-induced PKB/Akt phosphorylation in the tumor cells using the reverse treatment schedule (Fig. 5, D and E)Citation . Most probably this might be due to a different mechanism for VEGFR versus ErbB1/2-receptor-mediated PI3K activation upstream of PKB/Akt. PI3K is directly recruited to the specific docking site of the VEGFR, and this complex might not immediately be affected on inhibition of the RTK activity once PI3K is bound to the receptor. On the other hand, ErbB1/2-mediated activation of PI3K requires a multistep signaling process via ErbB3 or ras and thus might be more sensitive to upstream inhibition of ErbB1/2 kinase. Mechanistic analysis could address these differences in further detail.

Activation of the VEGFR and ErbB Receptor by IR.
To demonstrate a direct activation of the specific RTKs in response to irradiation, the activity status of the ErbB receptor and the VEGFR was analyzed in A431 cells and in HUVECs, respectively. Detection of ErbB receptor phosphorylation was performed with a Tyr1086 site-specific antiphospho-ErbB1 antibody in whole cellular extracts. An increase of the phosphorylation level was already observed at 1 and 2 min after irradiation with 5 Gy, and a minimal shift of a slower migrating band could be observed, most probably a hyperphosphorylated form of the receptor (Fig. 6)Citation .



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  		Fig. 6. Ionizing radiation activates the ErbB1 receptor in tumor cells. After 24 h of serum deprivation, A431 cells were irradiated with 5 Gy and analyzed at the indicated time points or after treatment with epithelial growth factor (50 ng/ml) as positive control. The phosphorylation level of ErbB1 receptors was determined in whole cell extracts using an anti-ErbB1 receptor phosphoTyr1086 site-specific antibody. Samples were reanalyzed with an antibody against ErbB1 receptor to ensure the loading of equal amounts of protein.

 
To determine activation of VEGFR-2 in response to irradiation, we performed immunohistochemical analysis of endothelial cells with confocal laser-scanning microscopy. Unfortunately and due to low detection quality, no Western blotting experiments with specific antibodies against the activated, phosphorylated form of the VEGFR-2 could be used for additional analysis of immunoprecipitates or whole cellular extracts from primary endothelial cells. HUVECs were serum starved for 3 h and irradiated with 2 Gy in the presence or absence of PTK787/ZK222584. Immunofluorescent labeling was performed with the Tyr996 site-specific anti-phospho-VEGFR-2 polyclonal antibody. In parallel the nuclei were stained with 4',6-diamidino-2-phenylindole to facilitate cellular localization. In comparison to control cells, irradiated cells displayed a markedly enhanced, almost continuous rim staining at the site of the plasma cell membrane. On the other hand, pretreatment of cells with PTK787/ZK222584 before irradiation resulted in a less pronounced rim staining with only a few dotted structures, similar to the staining of control cells (Fig. 7)Citation . Together with the data showing RTK inhibitor-dependent control of IR-induced PKB/Akt phosphorylation, these results demonstrate VEGFR-mediated activation of the PI3K/Akt pathway in endothelial cells and ErbB receptor-dependent activation of this survival pathway in the A431 tumor cells (Fig. 8)Citation .



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  		Fig. 7. Ionizing radiation activates the vascular epithelial growth factor receptor (VEGFR) in human endothelial cells. Human umbilical vascular endothelial cells were serum deprived for 3 h in the absence or presence of 500 nM PTK787/ZK222584 and then irradiated with 2 Gy. The cells were fixed 5 min after treatment, and immunofluorescence labeling was performed with the rabbit polyclonal antibody anti-VEGFR-2-phosphoTyr996 site specific antibody. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The cells were subsequently examined by confocal laser-scanning microscopy. The experiments were repeated at least three times and representative images are displayed.

 


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  		Fig. 8. Scheme of the differential phosphatidylinositol 3'-kinase (PI3K)/Akt pathway activation in tumor and endothelial cells by ionizing radiation. Ionizing radiation dependent PI3K/Akt pathway activation is mediated by ErbB receptors in tumor cells and vascular epithelial growth factor receptors (VEGFRs) in endothelial cells, subsequently promoting cell survival. Ligand-specific activation is represented by dotted arrows. Ionizing radiation (IR)-specific activation is represented by solid arrows.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The relevance of the PKB/Akt protein kinase for cellular survival has been investigated in different cell types and in response to many stress factors (47, 48, 49, 50) . Radiation-induced PKB/Akt activation in endothelial cells has previously been demonstrated to contribute to an increase in radioresistance both in vitro and in vivo; however, the upstream molecular entities leading to an activated PI3K/Akt pathway have not been addressed thus far in endothelial cells (42) . In this article we have compared RTK dependence of radiation-induced PI3K/Akt activation in tumor cells with the activation pattern in endothelial cells using clinically relevant inhibitors of the ErbB1/2 class receptors and the VEGFR. Both growth factor ligands EGF and VEGF stimulated PKB/Akt phosphorylation through their corresponding growth factor receptors, which could be abrogated by the RTK inhibitors PKI166 and PTK787/ZK222584, respectively, indicating an intact growth factor-induced signaling network from the RTKs to PKB/Akt in endothelial cells. Interestingly, IR dose and time-dependent activation of PKB/Akt in these cells closely followed the VEGF- but not the EGF-mediated activation pattern of this survival pathway. Radiation-induced PKB/Akt phosphorylation was strongly down-regulated by the VEGF-RTK inhibitor in the endothelial cells, whereas the ErbB receptor inhibitor did not decrease IR-induced PKB/Akt-stimulation in these cells. An opposite receptor dependence for radiation-induced PKB/Akt-phosphorylation was observed in the tumor cell line A431, similar to previously observed ErbB-receptor mediated activation of PKB/Akt in response to IR in tumor cells. Specific VEGF-independent but VEGFR-mediated PKB/Akt-activation was additionally corroborated through the direct activation of the PTK787/ZK222584-sensitive VEGFR-2 by IR.

Using PKI166 and PTK787/ZK222584, we could selectively discriminate between ErbB1/2- and VEGFR protein tyrosine kinase-mediated PKB/Akt phosphorylation in response to IR. Both inhibitors were intensively characterized with regard to their in vitro and in vivo specificity (35 , 51) , and especially PTK787/ZK222584 displays high VEGFR-specific inhibitory activity at the concentration used in this article (Ref. 35 and J. Wood, personal communication). Nevertheless, we cannot exclude that other kinases are (co-)activated by IR, coinhibited by PTK787/ZK222584, and at the same time contribute to IR-induced PKB/Akt-phosphorylation. On the other hand, the signaling pathway linking an activated VEGF-receptor with PI3K/Akt is well established in endothelial cells. More importantly, we demonstrated a differential mechanism for PKB/Akt activation in endothelial and tumor cells in response to irradiation and pinpoint to a responsible RTK activity and a relevant inhibitor. This is important with regard to the establishment of novel combined treatment modalities because growth factor-independent but IR-induced RTK-dependent PKB/Akt activation will also contribute to the resistance of irradiated vascular endothelium (52) .

Both enhanced cell proliferation and DNA repair as well as reduced apoptosis are part of a cytoprotective response and are induced in response to growth factor ligand- and IR-activated RTKs. Primary IR-induced RTK activation has been intensively investigated for ErbB receptor tyrosine kinases in tumor cells; however, the exact mechanism for their activation is still unclear (45 , 53) . The generation of reactive oxygen and nitrogen species may shift the steady-state tyrosine phosphorylation status of RTKs to its phosphorylated active form due to the deactivation of critical cysteine residues in the catalytic center of corresponding protein phosphatases (54, 55, 56, 57, 58, 59) . On the other hand, reactive oxygen species also affect lipid bilayer composition and rigidity, which might ultimately lead to a proximity effect and transphosphorylation of plasma membrane integrated RTKs (60) . Interestingly, although ligand-independent, IR-induced RTK (auto-)transphosphorylation is induced to a lower level in comparison to ligand-mediated RTK activation, downstream PKB/Akt phosphorylation is induced to a similar extent by IR and the corresponding growth factor (46 , 61 , 62) . We also observed a lower level of IR-induced VEGFR activation in endothelial cells, but again, the level of PKB/Akt phosphorylation was enhanced to a similar extent by IR and VEGF. Activation of multiple ErbB receptor isoforms in parallel might explain that low level of RTK phosphorylation on the individual isoform level is still sufficient to induce strong downstream signaling in a cumulative way or to overcome a signaling threshold. This might also apply to the low level of receptor phosphorylation of the different VEGFR isoforms, which could be determined by Western blotting in response to IR (data not shown). More importantly, IR-induced site-specific VEGFR-2 phosphorylation could be detected by confocal microscopy on the individual cellular level and could be abrogated by pretreatment with the VEGFR inhibitor PTK787/ZK222584.

Ligand-dependent activation of RTKs are mainly determined by the affinity of the ligand to the corresponding receptor-isoform. In-depth analysis by Dent et al. on the level of ErbB receptors revealed that IR-induced RTK activation does not discriminate between the different ErbB receptor isoforms and their different ligand affinities (61) . Rather, it is the expression level of the isoforms that predetermines IR-mediated RTK isoform activation. These studies have mainly been performed in tumor cells in which ErbB-mediated signaling dominate proliferation and survival processes not least because of a high expression level of these RTKs. We used primary, not established, proliferating endothelial cells for these studies, which most probably resemble in vivo endothelial cell structures with regard to RTK expression. Thus, the differential RTK dependence of IR-induced PKB/Akt phosphorylation might be due to a shifted RTK expression profile toward elevated VEGFRs in endothelial cells in comparison to ErbB receptor overexpression in ErbB receptor-dominated tumor cells or due to a change in the relative importance of a specific pathway. However, we cannot exclude a distinct activation of VEGFRs by IR, which might be linked to inter- and intramolecular cross-talks between the different VEGFR isoforms or the release of trapped signaling agonists such as VEGF or PIGF (63) . However, our irradiation experiments performed in presence of trapping anti-VEGF antibodies do not support such a scenario. A detailed quantification and analysis of PI3K recruitment to the different VEGFR isoforms will further clarify the mechanism of RTK-dependence for PI3K/Akt pathway activation in response to irradiation.

IR is a very powerful therapy against cancer because of its cytotoxicity mainly induced by DNA damage. However, IR also induces stress responses promoting cell survival and repopulation eventually leading to treatment resistance. Activation of the ErbB receptors followed by downstream activation of the PI3K/Akt pathway upon IR exposure was proposed to be a central step favoring tumor cell survival and proliferation. Moreover, this mechanism was shown to stimulate ErbB-receptor mediated VEGF expression from tumor cells indirectly promoting an angiogenic response or at least an antiapoptotic stimulus, which further contributes to enhanced radioresistance.

However, irradiation targets both tumor and endothelial cells, and interestingly, the radiosensitivity of the tumor microvasculature and microvascular damage strongly contributes to the tumor response to radiation (64) . Thus, targeting an intrinsic treatment threshold in endothelial cells of the tumor vasculature sensitizes the tumor for IR. Besides the cytotoxic effect of IR on the endothelial cell level, a direct cytoprotective stress response is induced by the activation of the PI3K/Akt pathway additionally promoting endothelial cell survival (41 , 51) . Consequently, PI3K inhibitors such as LY294002 or wortmanin increase IR-induced cytotoxicity in endothelial cells and also enhance radiation-induced tumor growth delay in animal tumor models. Unfortunately, these inhibitors are overly toxic for clinical studies.

Other preclinical studies demonstrated that specific inhibitors of angiogenesis such as neutralizing antihuman VEGF165 antibodies and anti-VEGFR-2 monoclonal antibodies enhance radiation-induced tumor growth control. In addition, many studies are performed in vitro and in vivo using small molecular pharmacological inhibitors of the VEGFR-2 tyrosine kinase, such as PTK787/ZK222548, in combination with IR and demonstrate a strongly potentiated effect of this combined treatment modality compared with each treatment modality alone (65, 66, 67, 68) . A combined treatment modality using anti-VEGF antibodies in combination with IR does induce an enhanced antiangiogenic effect by decreasing the growth and survival promoting effect mediated by the growth factor ligand VEGF. On the other hand, a specific clinically relevant VEGFR tyrosine kinase inhibitor will additionally abrogate direct IR-induced and receptor-mediated signaling in the endothelial tumor compartment, further contributing to a cooperative antitumoral effect of IR in combination with inhibitors of angiogenesis.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Hoechli from the service center for microscopical structure analysis (University of Zurich) for help in running the confocal microscopy experiments. We also thank Peter Traxler and Jeanette Wood (Novartis-Pharma) for the kind gift of PKI166 and PTK787/ZK222584.


   FOOTNOTES
 
Grant support: Swiss Cancer League (D. Zingg, M. Pruschy), the Radium Fonds, Hartmann Müller-Foundation and Stiftung zur Krebsbekämpfung (O. Riesterer) and an EORTC-Astra Zeneca Translational Research Fellowship (M. Pruschy).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: D. Zingg and O. Riesterer contributed equally to this work.

Requests for reprints: Martin Pruschy, Laboratory for Molecular Radiobiology, Department of Radiation Oncology, University Hospital Zurich, Ramistr. 100, CH-8091 Zurich, Switzerland. Phone: 411-255-8549; Fax: 411-255-44-35; E-mail: martin.pruschy{at}usz.ch

Received 10/28/03. Revised 4/21/04. Accepted 5/24/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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