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Cancer Research 68, 1691, March 15, 2008. doi: 10.1158/0008-5472.CAN-07-2372
© 2008 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

EphA2 Is an Essential Mediator of UV Radiation–Induced Apoptosis

Guoqi Zhang1,2, Ching-Ni Njauw1, Jong Min Park1, Chie Naruse4, Masahide Asano4 and Hensin Tsao1,2,3

1 Wellman Center for Photomedicine, 2 Department of Dermatology, Harvard Medical School, and 3 MGH Cancer Center, Massachusetts General Hospital, Boston, Massachusetts; and 4 Division of Transgenic Animal Science, Advanced Science Research Center, Kanazawa University, Kanazawa, Japan

Requests for reprints: Hensin Tsao, Department of Dermatology, Massachusetts General Hospital, Bartlett 622, 48 Blossom Street, Boston, MA 02114. Phone: 617-726-9569; Fax: 617-724-2745; E-mail: htsao{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
One of the physiologic consequences of excessive UV radiation (UVR) exposure is apoptosis. This critical response serves to eliminate genetically injured cells and arises, in part, from activation of DNA damage and p53 signaling. Other contributory pathways, however, likely exist but have not been fully characterized. In a recent global screen of UVR response genes in melanocytes, we identified the receptor tyrosine kinase EPHA2. Using a combination of genetic and pharmacologic approaches, we set out to investigate the upstream regulation of EphA2 by UVR and the functional consequences of this effect. We found that the UVR-associated increase in EphA2 occurs in melanocytes, keratinocytes, and fibroblasts from both human and murine sources. More specifically, UVR effectively up-regulated EphA2 individually in p53-null, p63-null, and p73-null murine embryonic fibroblasts (MEF), suggesting that the p53 family of transcription factors is not essential for the observed effect. However, inhibition of mitogen-activated protein kinase (MAPK) signaling by U0126 and PD98059 significantly reduced the UVR response whereas overexpression of oncogenic NRAS led to an increase in EphA2. These results confirm that UVR induces EphA2 by a p53-independent, but MAPK-dependent, mechanism. In response to UV irradiation, Epha2–/– MEFs were highly resistant to UVR-mediated cytotoxicity and apoptosis whereas introduction of EphA2 into both wild-type and p53-null MEFs led to activation of an apoptotic program that can be blocked by caspase-8 inhibition. These functional findings suggest that EphA2 is in fact an essential p53-independent, caspase-8–dependent proapoptotic factor induced by UVR. [Cancer Res 2008;68(6):1691–6]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The orchestrated genetic and biochemical response of various cells to UV radiation (UVR) is highly complex and is still largely unresolved. Much of the intracellular signaling and transcriptional events are aimed at defending the genome against mutagenic stress brought about by excessive UVR. In addition, reactive oxygen species contributes to cellular stress and triggers intracellular signaling and DNA damage (1). The p53 transcription factor has drawn particular attention given its central role in DNA damage and apoptotic signaling. However, UVR-induced apoptosis is highly complex and seems to occur through both p53-dependent and p53-independent pathways (2).

In a recent effort to catalogue all UV-mediated transcriptional events in melanocytes, we identified a p53 transcriptional target, EPHA2, which is significantly up-regulated by UVR (3). EphA2 belongs to the Eph receptor tyrosine kinase (RTK) family, which is the largest group of tyrosine kinases in the genome (4). The Eph receptors mediate cell-cell signaling through glycosylphosphatidylinositol lipid-anchored ligands (ephrin A) or transmembrane ligands (ephrin B). Because high EphA2 levels have been reported in melanomas (5, 6) and have been associated with a more malignant phenotype (7), the induction of EphA2 by UVR exposure provides a possible mechanistic link between excessive UV exposure and melanoma risk. Moreover, a large body of evidence suggests that EphA2 is overexpressed in many cancer types (8) thereby raising the possibility that EphA2 controls basic cellular processes that are not limited to pigment cells. We thus set out to further characterize the pathway(s) responsible for the up-regulation of EphA2 by UVR and the possible role that this gene plays in UV photobiology.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies and inhibitors. Antibodies used in this study were as follows: polyclonal anti-EphA2(C-20), monoclonal anti–β-tubulin (D-10), polyclonal anti-p63 and polyclonal anti-p73 (Santa Cruz); monoclonal anti–glyceraldehyde-3-phosphate dehydrogenase (Abcam); polyclonal anti-p53 (CM5; Novocastra Laboratories Ltd.); polyclonal anti–extracellular signal–regulated kinase (ERK), anti–phospho-ERK (Thr202/204), and rabbit monoclonal anti–poly(ADP-ribose) polymerase (PARP; Cell Signaling Technology); goat anti-mouse conjugated horseradish peroxidase (HRP; Bio-Rad); and goat anti-rabbit IgG-HRP (A1905; Santa Cruz).

Pharmacologic inhibitors of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK; U0126 and PD98059), p38 MAPK inhibitor [2-(4-chlorophenyl)-4-(4-fluorophenyl)-5-pyridin-4-yl-1,2-dihydropyrazol-3-one], as well as c-jun NH2-terminal kinase (JNK) inhibitor (JNK Inhibitor II; SP600125) were all purchased from Calbiochem. Phosphatidylinositol-3-kinase inhibitor (LY294002) was purchased from Cell Signaling Technology, Inc. Both caspase-8 and caspase-9 inhibitors were from Calbiochem.

Cell culture and UV irradiation. Normal human melanocytes (NHM) were purchased from Cascade Biologics, whereas immortalized NHMs and immortalized normal human keratinocytes (NHK; both engineered through the successive introduction of p53DD, CDK4R24C, and hTERT) were kindly provided by David Fisher (Dana-Farber Cancer Institute, Boston, MA) and James Rheinwald (Brigham and Women's Hospital, Boston, MA), respectively. Human melanoma cells lines have previously been published (9). Murine embryonic fibroblasts (MEF) from Trp53-, Trp63-, and Trp73-deficient mice were kindly provided by Elsa R. Flores (M. D. Anderson Cancer Center, Houston, TX), whereas MEFs from Epha2–/– mice (10) were isolated following standard protocols.

The NHMs and immortalized NHMs were maintained in Medium-254 (Cascade Biologics) supplemented with human melanocyte growth supplement and 1% penicillin/streptomycin. Melanoma cell lines and MEFs were maintained in DMEM supplemented with 10% fetal bovine serum (Sigma-Aldrich) containing 1% penicillin/streptomycin. Immortalized NHKs were cultured in keratinocyte serum-free medium (ker-sfm, Life Technologies, Inc./Invitrogen) supplemented with bovine pituitary extract (25 µg/mL; Life Technologies/Invitrogen), epidermal growth factor (0.2 ng/mL; Life Technologies/Invitrogen), and CaCl2 (0.4 mmol/L; Sigma)..

UV irradiation and treatment with inhibitors. We have previously described our UVB irradiation protocol (3). For inhibition studies, cells were plated the day before exposure and pretreated with molecular inhibitors targeting MEK, p38 MAPK, JNK, or phosphatidylinositol 3-kinase for 1 hour before irradiation. After UVB exposure, cells were then dislodged into 1x DPBS, centrifuged at 1,500 rpm for 5 minutes at 4°C, lysed, and frozen at –80°C until immunoblotting.

Immunoblot analysis. SDS-PAGE was done according to standard protocols. Ten micrograms of total cell lysate were diluted in 6x Laemmli buffer (Boston Bioproducts, Inc.) and loaded onto 10% precast gels (Bio-Rad), transferred onto Immobilon-P membrane (Millipore Corporation), and blocked with 5% nonfat milk (Bio-Rad) in Tween 0.1%/TBS. Primary antibody dilutions used in this study were as follows: polyclonal anti-EphA2(1:300), polyclonal anti-ERK (1:800), polyclonal anti–phospho-ERK (1:200), and polyclonal anti-p53, anti-p63, or anti-p73 (all 1:500). Loading equivalence was monitored with monoclonal anti–β-tubulin (1:150) or monoclonal anti-GADPH (1:3,500) and gels were visualized by enhanced chemiluminescence (Amersham Bioscience) after application of 1:5,000 goat anti-rabbit-HRP (1:5,000).

Plasmid construction and transfection. To generate the eukaryotic expression plasmids encoding wild-type (WT) NRAS and NRASQ61R, NRAS cDNAs were PCR cloned from the melanoma lines A375 and Roth (9), respectively. A donor human EPHA2 cDNA plasmid (Harvard Institute of Proteomics) was transferred into the pLP-IRESneo acceptor using the Creator kit (Clontech) per manufacturer's protocol. All plasmids were confirmed by DNA sequencing and immunoblotting after either transfection with FuGENE-6 (Roche Diagnostics) or nucleofection (Amaxa).

Apoptosis assays. For both UVB-associated and EphA2-mediated apoptosis, we plated 100,000 cells in 60-mm dishes ~21 hours before either UVB irradiation (80 mJ/cm2 UVB) or transfection (5 µg of pLP-IRES-EphA2 or pLP-IRES vectors). Treated cells were analyzed for intracellular caspase-3 activity at 24 hours (UVB irradiation) or 30 hours (EphA2 transfection) using the Caspase-3-Activity Detection Kit (Upstate).

For propidium iodide staining, 800,000 cells were harvested and then fixed with 70% ice-cold ethanol overnight. After fixation, cells were washed with PBS and then stained with 100 µg/mL propidium iodide in PBS containing 100 µg/mL RNase A and 0.1% NP40 (all from Sigma). Propidium iodide–stained cells were analyzed by FACSCalibur (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
UVR-mediated up-regulation of EphA2. To determine if UVR selectively induces EphA2 among other members of the Eph/ephrin family, we reanalyzed our microarray data (3) to assess the effect of UVR exposure on other ephrins and Eph receptors. As shown in Supplementary Fig. S1, EphA2 stands out among other Eph/ephrin genes in both level of stimulation and significance of increase. There is no apparent predilection for either Ephrins or Eph receptors, as a group, to be induced. Thus, EphA2, among its relatives, is selectively up-regulated by UVR.

Our previous data showed an increase in EphA2 mRNA levels in response to UVR (3). Figure 1A shows that EphA2 protein levels are induced by UVR within 5 hours and reach a maximum by ~12 hours postirradiation in NHMs; this level is sustained for at least 48 hours (data not shown). There is also a dose-dependent increase in EphA2 levels starting at ~15 mJ/cm2 UVB (Fig. 1B) and reaching a maximum of ~35 to 50 mJ/cm2.


Figure 1
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  		Figure 1. UV-mediated induction of EphA2 is cell autonomous and p53 independent. EphA2 induction by UVR in NHMs is dependent on time (A; 0, 0.25, 0.5, 2, 9, 12, and 24 h) and dose (B; 0, 15, 25, 35, and 50 mJ/cm2). The up-regulation of EphA2 is also observed in primary human melanocytes immortalized with p53DD, CDK4R24C, and hTERT (data not shown). Because immortalized NHMs have ectopically abrogated p53 function (by p53DD), we also set out to determine if melanoma lines were UVR responsive. C, NHMs and immortalized NHMs and NHKs (p53 inactivated by p53DD construct) all show induction of EphA2 by UVR. Bottom, eight melanoma cell lines (lane 1, SK-Mel 28; lane 2, SK Mel-119; lane 3, WM35; lane 4, MM455; lane 5, A375; lane 6, UACC903; lane 7, MM-LH; lane 8, WM164) were also subjected to UVB irradiation and exhibited a range of EphA2 inducible levels. Because the two cell lines with the greatest degree of up-regulation (lanes 7 and 8) both harbor homozygous p53 point mutations, these data suggest that intact p53 function is not needed for EphA2 induction in melanocytic systems. Although not directly mutated, p53 is presumed to be functionally crippled in cell lines 2 to 6 in which ARF is deleted ({Delta}ARF). Similar results were obtained with primary human keratinocytes. D, we irradiated NIH-3T3 cells, WT MEFs, and Cdkn2a–/– MEFs and found that the EphA2 response was preserved in these cells. To determine if the p53 family of transcription factors is required for this observed effect, we subjected MEFs deficient in p53, p63, and p73 to UVB and found that EphA2 is appropriately elevated in these lines.

 
Because there are extant reports that EphA2 can be transcriptionally induced by p53, we used two complementary strategies to determine if the UVR-mediated up-regulation of EphA2 is dependent on p53 in melanocytes. First, we exposed NHMs that have been immortalized with p53DD/CDK4R24C/hTERT to UVR and found a similar level of EphA2 induction as the nonimmortalized NHMs (Fig. 1C). Next, we irradiated a panel of melanoma cell lines and found that lines with homozygous deleterious p53 mutations (WM164, p.Tyr220Cys; MM-LH, p.Gly244Arg) were still able to up-regulate EphA2 in response to UVR (Fig. 1C). These initial results provide suggestive evidence that primary immortalized and transformed human melanocytes do not require p53 for the observed UVR EphA2 effect.

Although we initially identified this EphA2 regulatory event in melanocytic systems, all cells maintain the capacity to counter extracellular stress, and thus we hypothesized that this novel UVR response may be preserved in cells of different origins. We subjected immortalized p53-inactivated normal human keratinocytes (i.e., by introduction of p53DD/CDK4R24C/hTERT) to UVR and found a similar level of EphA2 induction as in melanocytes (Fig. 1C). This is significant because keratinocytes represent the most direct target of both UVR stress and possible p53 mutagenesis (11).

Additionally, MEFs represent a highly tractable system to monitor physiologic stress in a definable genetic context. As shown in Fig. 1D, irradiation of WT MEFs, NIH-3T3, cells and Cdkn2a–/– MEFs all reproducibly resulted in EphA2 increases. The ability to introduce MEFs into the analysis also allowed us to more definitively exclude the p53 family of transcription factors. To this end, we subjected MEFs that were null for Trp53, Trp63, and Trp73 to UVR and found that EphA2 protein levels were in fact appropriately induced in all of these MEFs (Fig. 1D). Taken together, our results using immortalized melanocytes, p53, and CDKN2A-ARF–inactivated melanoma cells and genetically defined MEFs all strongly support the contention that p53, p63, and p73 are not individually necessary for the observed EphA2 regulation, although we cannot eliminate the possibility that there is functional compensation between the family members. It is worth mentioning, however, that these results do not contradict earlier findings that EphA2 is a p53 target gene (12); in fact, our data provide evidence for a higher order of complexity in EphA2 response to cellular UVR stress.

Beyond regulation by the p53 family of transcription factors, recent studies have found that EphA2 is also a direct transcriptional target of the Ras/Raf/MAPK signaling (13); thus, we surmised that this cascade may be an alternative mechanism to increase EphA2. To specifically eliminate all potential contributions from p53, we carried out our studies using MEFs lacking p53 function (Cdkn2a–/– or Trp53–/–). As shown in Fig. 2A , preincubation of MEFs with MEK inhibitor PD98059 (Fig. 2A) or U0126 at increasing concentrations (Fig. 2B) dramatically reduced the UVR-mediated increases in EphA2. Exposure of cells to p38 MAPK and JNK inhibitors did not show the same inhibitory effect (Fig. 2C). Interestingly, we did observe, on occasion, higher EphA2 induction when p38 MAPK or JNK was inhibited thereby pointing to possible counterregulation within the UVR stress–activated MAPK response.


Figure 2
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  		Figure 2. EphA2 induction by UVR is dependent on MAPK signaling. Cells were treated with MEK inhibitors PD98059 (50 µmol/L; A) or U0126 (dosed 3, 8, or 14 µmol/L; B) for 1 h before UVB exposure (40 mJ/cm2); EphA2 levels were determined 24 h after irradiation. The relative level of EphA2 in unirradiated cells is normalized to 1.0. There is a significant reduction in UVR-mediated up-regulation of EphA2 although it is not always complete. C, the induction of EphA2 at 24 h by UVB is not inhibited by 3.5 µmol/L of p38 MAPK inhibitor, 5.7 µmol/L of JNK inhibitor II, or 10 µmol/L of LY294002 (a phosphatidylinositol 3-kinase inhibitor). D, EphA2 can be induced by an oncogenic Nras (NrasQ61R; N*) but not by WT Nras (N) or vector (V).

 
To further corroborate that Ras/MAPK signaling is involved in EphA2 regulation, we ectopically expressed oncogenic NRAS (NrasQ61R; Fig. 2D) in NHMs and reproduced the increase in EphA2. One can thus conclude that the UVR-mediated stimulation of MAPK signaling is the mechanism responsible, at least in part, for the observed EphA2 induction.

Taken together, we show for the first time that a cancer-associated RTK, EphA2, is up-regulated by UVR in a p53-independent, but MAPK-dependent, fashion. This finding is provocative given the high frequency of both oncogene-associated activation of MAPK signaling and the mutational inactivation of p53 in cancer. Thus, to begin understanding the functional consequences of this increase in EphA2 by UVR, we set out to assess a common physiology shared by UVR exposure and cancer cell survival.

UVR-induced apoptosis is dependent on EphA2. We undertook a genetic approach to abrogate the EphA2 response. More specifically, we exposed both WT and primary nonimmortalized MEFs from Epha2–/– mice to UVR and determined the effect on cell survival. As expected, there was a complete absence of the UVR-mediated EphA2 induction in the Epha2–/– MEFs (data not shown). Strikingly, the Epha2-null primary MEFs showed substantial resistance to UVR-mediated cytotoxicity (Fig. 3A ). This observation lends itself to the hypothesis that EphA2 is in fact an essential modulator of UVR-associated apoptosis. We found that primary Epha2-null MEFs exposed to UVB (80 mJ/cm2) exhibited a significantly reduced level of caspase-3 activity and DNA fragmentation (i.e., sub-G1 fraction) compared with WT MEFs (Fig. 3B and C). The Epha2–/– MEFs were nevertheless vulnerable to Adriamycin-induced apoptosis (data not shown).


Figure 3
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  		Figure 3. Epha2–/– MEFs are more resistant to UV-induced cytotoxicity and UV-mediated apoptosis. A, WT and Epha2-deficient MEFs were exposed to increasing doses of UVB and cell survival was measured at 24 h. Epha2–/– MEFs were clearly more resistant to UVR cytotoxicity (*, P < .05; **, P < .001). The apoptotic response to UVR, as measured by caspase-3 activity level (B) and DNA fragmentation (C), was preserved in WT MEFs but dramatically reduced in Epha2–/– MEFs. Epha2–/– MEFs exposed to Adriamycin exhibited normal apoptosis (data not shown).

 
EphA2 induces apoptosis via caspase-8–dependent signaling. One explanation for the observed UVR findings is that up-regulation of EphA2 directly induces apoptosis. To test this possibility, we overexpressed EphA2 in p53-null human melanocytes (i.e., immortalized NHMs), WT MEFs, and p53-null MEFs and observed strong increases in caspase-3 activity (Fig. 4A ) in all three cell types and cleavage of PARP in the melanocytes (Fig. 4B). It thus seems that p53 is not essential for either the induction of EphA2 by UVR or the execution of the EphA2-associated apoptotic program. To further elucidate whether EphA2 signals through the intrinsic or extrinsic pathway, we treated immortalized NHM with specific caspase-8 and caspase-9 inhibitors (both at 20 µmol/L). As shown in Fig. 4C, there is selective abrogation of the EphA2-mediated PARP cleavage when caspase-8 activity is inhibited, suggesting that EphA2 engages the extrinsic pathway to execute its death program.


Figure 4
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  		Figure 4. Ectopic overexpression of EphA2 induces apoptosis in a p53-independent, caspase-8–dependent manner. A, because loss of EphA2 compromises UVR-mediated apoptosis, we set out to determine if overexpression of EphA2 can induce apoptosis. Ectopic expression of EphA2 led to an increase in apoptosis in immortalized NHMs (inactivated by dominant negative p53DD), WT MEFs, and p53–/– MEFs. B, EphA2 induces cleavage of PARP in immortalized NHMs. These results suggest that EphA2 does not depend on intact p53 to bring about apoptosis. C, immortalized NHMs transfected with either vector (V) or EphA2 (E) and treated with specific caspase-8 (C8i) or caspase-9 (C9i) inhibitors. There is selective inhibition of PARP cleavage by EphA2 when caspase-8, but not caspase-9, activity is abrogated, suggesting that EphA2-mediated effects depend, in part, on caspase-8 activity. D, model of UV-EphA2 circuitry (see text).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In summary, the major finding of our present study is that EphA2 seems to be an essential p53-independent, but MEK-dependent, mediator of UVR-induced apoptosis. The results support a model (Fig. 4D) whereby UVR exposure activates the Ras/MAPK signaling cascade, which in turn leads to an increase in EphA2 levels and a triggering of the apoptotic program. Because p53 is known to be induced both by UVR and a transcriptional regulator of EphA2 (12, 14), it is possible that both p53 and Ras/MAPK signals resulting from UVR exposure converge on EphA2. From our data, it seems that downstream triggering of apoptosis by EphA2 is independent of p53 but critically dependent on caspase-8, a component of the extrinsic pathway. It is not possible to fully exclude participation by the intrinsic pathway because there may be distinct triggering mechanisms in different cell types; studies to uncover these mechanistic details are currently under way.

Our findings relating EphA2 to UVR are consonant with emerging data to suggest that increases in EphA2 levels may serve as an adaptive response to various forms of microenvironmental stress. Xu et al. (15) recently reported that EphA2 levels were induced by hypertonic stress in murine kidney cells derived from the inner medullary collecting duct. Similarly, Baldwin et al. (16) found that renal ischemic-reperfusion injury also led to increased EphA2 levels through both ERK and Src kinases. Finally, Li et al. found that colonic carcinoma cells exposed to deoxycholate led to an increase in EphA2 by a MAPK-dependent, but p53-independent, mechanism. With the availability of specific Epha2–/– cells, our studies enrich this collective experience by directly implicating EphA2 in the apoptotic program attendant to UVR stress.

The role of EphA2 in tumor physiology is well described but highly complex. In epidermal keratinocytes, EphA2 seems to harbor tumor-suppressive effects. Normal epidermal keratinocytes express high levels of both EphA1 and EphA2 (5, 17) but sequentially lose expression of EphA1 during squamous cell carcinoma development. Moreover, Guo et al. (18) recently showed that mice deficient for Epha2 exhibit a greater susceptibility to squamous cell carcinoma tumorigenesis compared with normal mice in the 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate skin carcinogenesis model. These findings in keratinocytic neoplasms stand in sharp contrast to other cancers such as melanomas, breast cancer, prostate cancer, non–small-cell lung cancer, and colon cancer where EphA2 has been shown to be frequently overexpressed (8), suggesting a more oncogenic role. In melanoma cells, for instance, high EphA2 expression correlates with a more aggressive phenotype characterized by "vasculogenic mimicry" (6, 19–21); this vasculogenic phenomenon is consistent with the known role of EphA2 in tumor angiogenesis (22, 23). Moreover, high levels of EphA2 are also associated with worse prognoses in ovarian cancers (24, 25), renal cell carcinomas (26), and esophageal squamous cell carcinomas (27). Taken together, these findings have led to some early studies suggesting that EphA2 may be a viable therapeutic target in cancer (22, 28).

The balance between the oncogenic and apoptotic effects of EphA2 likely depends on the cellular context and the unique pathways that are activated in response to various microenvironmental stressors. This has been observed with other canonical oncogenes including c-Myc (29) and Ras (30). Clearly, precise regulation of the protumorigenic and proapoptotic signals is critical for the life and death decisions confronting normal cells under stress and transformed cells under selection. Although the mechanisms that explain the seemingly paradoxical effects of EphA2 are unknown, there are some hypothetical models. In a two-signal model, EphA2 may induce apoptosis and proliferation/survival signals through distinct pathways; in cancers, the proapoptotic pathway may be abrogated by concomitant mutations. Alternatively, in a gain-of-resistance model, cells that are selected to survive in the face of escalating death stimuli, including EphA2, may become genetically more aggressive. Additional investigations are clearly needed to fully appreciate the cellular and genetic contexts that specify EphA2 function.


   Acknowledgments
 
Grant support: This work was supported in part by the Department of Defense (H. Tsao) and by the generous contributions of philanthropic donors to Massachusetts General Hospital.

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.

We thank Martin Purschke for assistance with the flow cytometry.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 6/25/07. Revised 10/10/07. Accepted 1/18/08.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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J. Wykosky and W. Debinski
The EphA2 Receptor and EphrinA1 Ligand in Solid Tumors: Function and Therapeutic Targeting
Mol. Cancer Res., December 1, 2008; 6(12): 1795 - 1806.
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