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Experimental Therapeutics |
Cancer Research Institute [M. A., C. C., M. M., F. M., W. M. K.], Division of Gastroenterology [W. M. K.], University of California, San Francisco, California 94143
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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CAR3
is a 46-kDa transmembrane protein that enables virus attachment via interaction with the adenovirus fiber-knob protein. In normal cells, CAR is associated with tight junction protein complexes (3)
. Loss of CAR expression on the cell surface significantly reduces infectivity, which can be rescued by ectopic expression of the protein. Subsequent to the attachment to CAR, internalization of adenovirus is mediated by binding of the virus penton base to 
vß3- and vß5-integrins (4)
. Recent reports demonstrate a frequent reduction of CAR expression in highly malignant tumors (5, 6, 7)
. Our own assessment of CAR expression in human colorectal, liver, and breast cancer confirms this observation.4
Despite the importance of CAR for adenovirus-based cancer therapies, information regarding molecular mechanisms regulating CAR expression in normal and malignant cells is not available. It has been well established that expression of the tight junction protein ZO-1 and the adherens junction protein E-cadherin is frequently lost in highly malignant tumors (8, 9, 10)
, and evidence suggests that this loss of expression can be mediated through oncogenic signaling, e.g., via the Ras-activated ERK signaling pathway (11)
. Conversely, restoration of ZO-1 expression at the cell surface after inhibition of MEK has been demonstrated (11)
. If CAR is similarly regulated, manipulation of the Raf-MEK-ERK pathway could be central to an anticancer strategy using adenovirus vectors.
In this study we investigated the effect of signaling through the Raf-MEK-ERK pathway on CAR expression in cell line models of human cancers (pancreatic and colorectal) that are potential targets for adenovirus-based therapies. We also used cells from a well-characterized mouse skin cancer progression model (12) . We show that pharmacological signaling through the Raf-MEK-ERK pathway up-regulates CAR expression on the cell surface of cancer cells, leading to increased uptake of adenovirus and cell killing.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RAF1:ER) have been described previously (13
, 14)
.
Anti-CAR Antibodies.
The mouse monoclonal anti-CAR antibody RmcB was a gift from Dr. J. Bergelson. The rabbit polyclonal antibodies Ab72 and Ab248 were gifts from Dr. L. Post (Onyx Pharmaceuticals, Inc., Richmond, CA). Ab72 was generated against recombinant full-length CAR protein, whereas Ab248 was made against a peptide representing the COOH terminus of CAR. The specificities of these polyclonal antibodies were documented by analyzing CHO-CAR cells by Western blot analysis and FACS. In addition, proteins were immunoprecipitated from colorectal cancer cells with use of the monoclonal antibody RmcB. Precipitated proteins were resolved by SDS-PAGE, and membranes were probed with Ab72 or Ab248, respectively. In both instances, 46-kDa protein bands were detected. Preimmune serum was available as a control and detected no discernable protein band (data not shown).
Signal Transduction Inhibitors.
For inhibition of Raf-MEK-ERK signaling, the MEK inhibitors U0126 and PD184352 (Calbiochem, San Diego, CA) were used at final concentrations of up to 20 µM. As a control, cells were treated with U0124 (Calbiochem), a compound that is a chemical analogue of U0126 without MEK-inhibitory activity (15)
. The PI3K inhibitor LY292004 (Calbiochem) was used at a final concentration of 40 µM.
Induction of Raf-1 in MDCK Cells Expressing the EGFP-RAF1:ER Fusion Protein.
Cells were grown on Permanox multiwell slides (Nalge Nunc, Naperville, IL). For the induction of Raf-1, cells were grown in medium containing 4-HT (1 µg/ml) or ethanol (as a control) for up to 72 h. Immunofluorescence staining was then performed using antibodies against CAR, ZO-1, and E-cadherin (see below).
Western Blot Analysis and Protein Coimmunoprecipitation.
For Western blot analysis, cells were washed with cold PBS and subsequently scraped into isotonic buffer [10 mM Tris (pH 7.4), 140 mM NaCl, 2 mM DTT, Complete Mini protease inhibitor (Boehringer, Mannheim, Germany)]. Cells were then disintegrated by passage through a 25-gauge syringe. Membrane and cytoplasmic fractions were subsequently separated by centrifugation at 100,000 x g and subjected to electrophoresis and protein immunoblotting. Membrane fractions were resuspended in isotonic buffer. Polyvinylidene difluoride membranes were probed with the anti-CAR antibodies Ab72 and Ab248 and antibodies against E-cadherin (BD Transduction Laboratories, Lexington, KY), ZO-1 (mouse anti-ZO1 and rabbit anti-ZO-1; Zymed, S. San Francisco, CA), Erk, and ß-actin (Sigma, St. Louis, MO). For immunoprecipitation experiments, HCT116 cells were scraped into lysis buffer [0.5% sodium deoxycholate, 1% NP40, 0.5% SDS, 0.15 M NaCl, 10 mM Tris (pH 7.5)]. For each precipitation, 300 µg of protein lysate were incubated overnight with anti-CAR antibody Ab248. Precipitation was performed with protein-A agarose beads. After the pellet was washed, precipitated proteins were recovered from the beads by boiling in sample buffer (NuPage LDS Sample Buffer; Invitrogen, San Diego, CA) for 5 min. Proteins were resolved by Western blot analysis (see above). Signals were visualized by enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ).
Immunofluorescence Microscopy.
For immunofluorescence staining, cells were grown on Permanox multiwell slides (Nalge Nunc). Cells were fixed with 1% paraformaldehyde for 2 min. After permeabilization with 0.3% Triton-X for 10 min and blocking with 2% BSA, sections were incubated with primary antibodies against CAR, E-cadherin, or ZO-1. Serotype-matched immunoglobulin from rabbits or mice was used as a control. Primary antibodies were detected with a FITC-conjugated antirabbit antibody or a Cy-3-conjugated antimouse antibody, respectively (Molecular Probes, Eugene, OR). Multicolor fluorescence microscopy was performed using a DMRCA fluorescence microscope (Leica, Wetzlar, Germany) equipped with an Orca-100 charge-coupled device camera (Hamamatsu, Bridgewater, NJ). For confocal fluorescence microscopy, we used a LSM 510 microscope equipped with a two-photon laser (Carl Zeiss, Jena Germany). Quantitative image analysis was performed with Openlab software (Improvision, Lexington, MA).
For each experimental condition, 10 representative digital images were obtained at a magnification of x400. Signal intensities were integrated for the whole image after digital density slicing and calculation of average signal intensities. The threshold for the density-slicing step was chosen in a way that only fluorescent signals at sites of cell-cell contact were measured, excluding cytoplasmic signals.
FACS Analysis of CAR and Adenovirus Protein Expression.
For CAR staining, cells were harvested after treatment with 0.25% trypsin. After being washed in PBS, cells were incubated with the anti-CAR antibody RmcB in binding buffer (PBS containing 2% BSA and 10% normal goat serum) at 4°C for 45 min. To stain for adenovirus hexon protein, cells were fixed in 1% paraformaldehyde, washed three times in PBS, and incubated in binding buffer containing a mouse antiadenovirus antiserum (Chemicon, Temecula, CA). After washing, cells were incubated with a Cy-3-conjugated antimouse antibody (Molecular Probes). Stained cells were analyzed on a FACSCalibur cytometer (Becton Dickinson, Franklin Lakes, NJ).
RNA Extraction and cDNA Synthesis.
Total RNA was extracted from cells and tissues with use of the RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized using 2.5 units/µl Moloney murine leukemia virus reverse transcriptase (Invitrogen). Reverse transcription was performed in a 100-µl final volume containing 500 ng of RNA template, 10 µl of 10x PCR Buffer, 30 µl of MgCl2 (25 mM), 4 µl of deoxynucleotide triphosphate mixture (25 mM each; all Roche Molecular Systems, Inc., Branchburg, NJ), 5 µl of Random Primers (100 µM; Invitrogen), and 1 µl of RNase inhibitor (Roche Molecular Systems). Reverse transcription reactions were incubated at room temperature for 10 min and at 48°C for 40 min, followed by 5 min at 95°C. cDNA was stored at -20°C until use.
Real-Time PCR.
For real-time PCR detection of CAR mRNA expression, oligonucleotide primers and TaqMan probe with the following sequences were used: forward, 5'-GGCGCTCCTGCTGTGC-3'; reverse, 5'-CTTTGGCTTTTTCAAT-CATCTCTTC-3'; probe, 5'-(6FAM)-TGCGGAGTAGTGGATTTCGCCAGAAG-(TAMRA)-3'. Cyclin D1 mRNA was detected using the following primers and TaqMan probe: forward, 5'-TACTACCGCCTCACACGCTTC-3'; reverse, 5'-TGCCAGGA-GCAGATCGAA-3'; probe, 5'-(6FAM)-ATCAAGTGTGACCCAGACTGCCTCCG-(TAMRA)-3'. PCR was conducted in triplicate with 50-µl reaction volumes of 1x PCR buffer A (Applied Biosystems, Foster City, CA), 2.5 mM MgCl2, 0.4 µM each primer, 200 µM each deoxynucleotide triphosphate, 100 nM probe, and 0.025 units/µl Taq Gold (Applied Biosystems). After the addition of 10 µl of primer/probe and 40 µl of cDNA (see above), PCR was performed using the following conditions: 1 cycle of 95°C for 12 min, followed by 40 cycles of 95°C for 20 s and 60°C for 1 min. Analyses were carried out using the sequence detection software supplied with the ABI 7700 (Applied Biosystems). Expression was quantified based on the changed in threshold cycle (-CT) for CAR expression relative to expression of ß-GUS.
Viruses and Viral Infection of Cells.
Ad5 is a human group C wild-type adenovirus, and dl1520 (ONYX-015) is a chimeric human group C adenovirus (Ad2 and Ad5) containing mutations of the E1B-55K and the E3B region (16)
The deletion of E1B-55K restricts replication of this virus to cells with disruption of the p53 tumor suppressor pathway (2
, 17)
. Wild-type D adenovirus harbors the same mutation of the E3 region as dl1520 but contains a wild-type E1B-55K region. dl312 is an E1A-deleted, replication-incompetent adenovirus (a gift from Dr. L. Johnson, Onyx Pharmaceuticals, Inc., Richmond, CA). All viruses were grown and titered on the human embryonic kidney cell line HEK293. Infections were performed as described previously (18)
. In brief, subconfluent cells were washed and incubated for 90 min at 37°C with infection medium containing 2% FBS and the amount of virus necessary to achieve the desired MOI. Subsequently, cells were washed with PBS, and fresh medium containing 10% FBS was added. For mock infections, cells were treated identically except that no virus was added.
Assessment of Infectivity with Adenovirus.
A nonreplicating, E1A-deleted adenovirus expressing EGFP (Ad-GFP) was used to measure the infectivity of cells. Infections were performed after 48 h of treatment with U0126 (10 µM), Ly294002 (40 µM), or DMSO, respectively. Cells were washed and then incubated in infection medium containing 2% FBS and Ad-GFP at a concentration suitable to achieve a MOI of 10, but no MEK or PI3K inhibitors. After 48 h, cells were trypsinized and washed with PBS. The percentage of GFP-expressing cells was determined by FACS analysis. To examine the specific role of the CAR-adenovirus interaction for adenovirus entry after signal transduction inhibition, cells were preincubated with recombinant adenovirus type 2 fiber (50 µg/ml; a gift from Dr. G. Nemerow, The Scripps Research Institute, La Jolla, CA), or Ad-GFP was preincubated with fusion protein of the extracellular domain of CAR and the Fc portion of human IgG (25 µg/ml; a gift from Dr. J. Bergelson). To rule out that these recombinant proteins were inhibiting adenovirus entry through nonspecific interactions with secondary (as yet unknown) cellular receptors, control cells were incubated with BSA (50 µg/ml).
Assessment of CPE.
HCT116 cells were treated with U0126 (10 µM) or DMSO for 48 h and subsequently infected with wild-type adenovirus (subtype 5), wild-type D, ONYX-015, or dl312, respectively, at MOIs of 0.1, 1.0, or 10, as described above. Remaining living cells were stained 48 h later with crystal violet, and low-magnification images were obtained. In addition, phase-contrast images of cells were obtained at a magnification of x400.
Virus Burst Assay.
HCT116 cells, treated with U0126 (10 µM) or DMSO for 48 h, were infected with wild-type adenovirus (Ad5) at a MOI of 10, and 48 h later were scraped into 2 ml of medium. Lysates were prepared by three cycles of freezing and thawing. HEK293 cells (human embryonic kidney cells expressing the E1 region of adenovirus type 2) were incubated with serial dilutions of the lysates. After 48 h, cells were harvested by trypsin treatment and stained for adenovirus proteins, as described above. FACS analysis was used to measure the fraction of cells positive for adenovirus protein, which allowed calculation of the number of viable virus particles in the cell lysates.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. At these concentrations, levels of total ERK remained mainly unchanged, which is in agreement with the published literature (Fig. 1A
; Ref. 15
). After treatment with the inhibitors, SW480 cells showed a significant increase in CAR protein expression, in particular in the membrane protein fraction (Fig. 1A)
, whereas control cells that were treated with either DMSO or U0124, an analogue of U0126 with no inhibitory effect on MEK1/2 (15)
, failed to induce any changes in CAR expression (Fig. 1A)
. Similar results were obtained in the human colorectal and pancreatic cancer cell lines HCT116 and MIA PaCa-2, respectively, as well as in the mouse skin cancer cells B9 (Fig. 1B)
.
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. Similarly, a reduction in protein levels in the same cells was found by immunofluorescence microscopic analysis (data not shown). These results suggest that the Raf-MEK-ERK and PI3K signal transduction pathways have differential effects on CAR expression, at least in this cell type.
Transcriptional Regulation of CAR Expression.
To assess whether the changes in CAR protein expression levels on inhibition of MEK resulted from changes in transcription of the CAR gene, we used a quantitative real-time PCR assay that allowed measurement of CAR relative to the expression levels of the housekeeping gene ß-GUS. In SW480 cells, CAR mRNA levels increased markedly after inhibition of MEK with U0126 (Fig. 2A)
. These findings were confirmed by Northern blotting (data not shown). To verify the effect of MEK inhibition on known downstream targets of the Raf-MEK-ERK pathway, we also analyzed RNA expression of the Cyclin D1 gene. As expected, Cyclin D1 RNA levels were reduced in SW480 cells treated with U0126 (Fig. 2B)
. Interestingly, RNA expression of both CAR and Cyclin D1 was reduced after treatment with the PI3K inhibitor LY292004. These data demonstrate that regulation of CAR expression by the Raf-MEK-ERK pathway is, at least in part, mediated at the transcriptional level. Furthermore, because both U0126 and LY292004 arrest cells in the G1 phase of the cell cycle (data not shown), these data demonstrate that the changes in CAR expression levels are not a result of cell cycle arrest but are specifically related to signaling through the Raf-MEK-ERK pathway.
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or PD184352 (data not shown). Treated SW480 cells showed CAR expression concentrated at cell-cell contacts, whereas the cytoplasmic staining was reduced and costaining for ZO-1 demonstrated a striking increase of ZO-1 protein at the cell surface (Fig. 3A)
. Confocal microscopy confirmed that both proteins localized at cell-cell contacts after treatment with MEK inhibitors (Fig. 3A)
. Similarly, CAR colocalized with the adherens junction protein E-cadherin (Fig. 3B)
. We also performed protein immunoprecipitation experiments to investigate whether CAR physically binds to these proteins. Although we did not detect an interaction with E-cadherin, we were able to coimmunoprecipitate ZO-1 with CAR (data not shown), confirming the published data on CAR-ZO-1 interaction in MDCK cells (21)
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50% (data not shown).
Reduced CAR Cell Surface Expression after Activation of Raf-1 in MDCK Cells Expressing the EGFP-RAF1:ER Fusion Protein.
We tested whether activation of the kinase upstream of MEK, Raf-1, is sufficient to decrease CAR cell surface expression. We used MDCK cells expressing the EGFP-RAF1:ER fusion protein, which becomes activated in the presence of tamoxifen (13)
. Monolayer cultures of these cells were treated with tamoxifen for 48 h. A significant increase of the phosphorylated form of ERK, as detected by Western blot analysis (data not shown), confirmed that the treatment activated the Raf-MEK-ERK pathway. Cells were immunostained with the anti-CAR antibody Ab248 and the mouse monoclonal anti-ZO-1 antibody. In the absence of activated Raf-1, a strong, consistent, and very homogeneous staining at the sites of cell-cell contact was detected for CAR and ZO-1 (Fig. 4A)
. In contrast, digital image analysis using digital density slicing revealed reduced signal intensity for CAR and ZO-1 after Raf-1 activation (Fig. 4B)
. For this measurement, fluorescent signals mainly at the sites of cell-cell contact were included. The cytoplasmic signal intensity was found to be at background levels before and after treatment with the inhibitor and was therefore not included in the measurement. The staining pattern for CAR was heterogeneous and partly interrupted after activation of Raf-1 (Fig. 4A)
. Furthermore, increased punctate staining was seen in the cytoplasm, whereas overall protein levels did not change, as determined by Western blot analysis (Fig. 4A
, and data not shown).
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. Infectivity of Colo 205 cells, in which no increase in CAR protein levels had been observed after MEK inhibition, remained at background levels before and after inhibitor treatment when cells were infected with Ad-GFP at a MOI of 10 (0.4 and 0.8%, respectively; data not shown). In additional experiments, we found increased expression of the early adenovirus protein E1A in cells infected with wild-type adenovirus after treatment with U0126 (data not shown). Infectivity with adenovirus was abolished in cells that had been preincubated with a recombinant adenovirus fiber protein (a gift from Dr. G. Nemerow), with or without treatment with MEK inhibitors (Fig. 5B)
. Similar results were obtained after incubation of Ad-GFP virus with a fusion protein of the extracellular domain of CAR with the Fc domain of human IgG (a gift of Dr. J. Bergelson) that blocks the CAR binding site of the adenovirus fiber-knob protein (Fig. 5B)
. These observations strongly suggest that the increased uptake of adenovirus after MEK inhibition results from up-regulation of CAR expression at the cell surface.
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). This effect was also seen after infection with the replication-selective virus ONYX-015 (dl1520; Fig. 6B
), whereas the nonreplicating dl312 virus mutant did not cause CPE (data not shown). Assessment of the yield of ONYX-015 from HCT116 cells by virus burst assay showed a 28-fold greater production of virus after MEK treatment compared with control cells (Fig. 6C)
. A similar increase in virus production after MEK inhibition was found for wild-type adenovirus (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Increasing evidence exists for a potential physiological role of CAR as a cell adhesion molecule. CAR forms homodimers, was found to physically interact with the tight-junction protein ZO-1, and participates in formation of the apical junction complex (21 , 23, 24, 25) . Because signaling through the Raf-MEK-ERK pathway has been found to down-regulate cell-cell adhesion complexes, e.g., tight- and adherens junctions (11) , we hypothesized that this pathway might also contribute to the down-regulation of CAR in cancer cells.
To test our hypothesis, we treated a set of gastrointestinal and skin cancer cell lines with the specific MEK inhibitors U0126 and PD184352, which led to increased levels of membrane-associated CAR protein (Fig. 1A)
. Examination of the subcellular localization of CAR after MEK inhibition revealed a striking increase in CAR protein expression at cell-cell junctions. Similarly, increased expression of the tight junction protein ZO-1 and the adherens junction protein E-cadherin was observed (Fig. 3)
. CAR colocalized with ZO-1, which is in agreement with the published report of a complex formation of both molecules MDCK cells (21)
. The up-regulation of CAR expression at sites of cell-cell contact was fully established after 24–48 h of treatment (Fig. 3B)
. This time course suggests that regulation of CAR by the Raf-MEK-ERK pathway is indirect and involves factors downstream of MEK. Regulation of CAR protein expression appears to be similar to that of other adherens junction proteins, notably E-cadherin and ß-catenin. In Ras-transformed MDCK cells, interruption of RAF-MEK-ERK signaling restored E-cadherin localization at adherens junctions (11)
. Also in MDCK cells, the MEK inhibitor PD098059 abolished disassembly of adherens junctions induced by treatment with hepatocyte growth factor/scatter factor (26)
.
In SW480 cells, the increase in CAR protein at the cell surface after inhibition of MEK was accompanied by an increase in CAR mRNA levels (Fig. 2)
. In contrast, CAR mRNA expression levels decreased after inhibition of another target of Ras signaling, PI3K, suggesting that these two signal transduction pathways downstream of Ras might have contrary effects on CAR expression. We therefore speculate that the relative activation of each signaling branch in different tumors results in different levels of CAR expression. Because inhibition of both Raf-MEK-ERK and Ras-PI3K signaling caused arrest of cells in the G1 phase of the cell cycle (accompanied by repression of Cyclin D1 gene expression), it is evident that the effect of Raf-MEK-ERK signaling on CAR expression is independent from the status of the cell cycle. In support of this conclusion, we observed no changes in CAR protein expression after treatment of SW480 cells with the cell cycle inhibitor mimosine, which arrests cells in G1 (data not shown). These observations are in agreement with the published finding that CAR expression is unchanged during the G1 phase of the cell cycle in A549 lung cancer cells (27)
.
In addition to transcriptional regulation of CAR, we found evidence for possible post-translational modifications of CAR protein after MEK inhibition: HCT116 cells demonstrated changes in the subcellular localization of CAR without significant changes in mRNA steady-state levels. It has already been demonstrated that fatty acid modification affects the subcellular localization of CAR (28) . However, it remains to be determined whether the Raf-1-MEK-ERK pathway regulates such modification.
We tested whether activation of Raf-1 signaling is sufficient to repress CAR expression, using MDCK cells that stably express a tamoxifen-inducible form of Raf-1 (14)
. Similar to previous reports demonstrating a dramatic down-regulation of expression of cell-cell adhesion molecules, including ZO-1 and E-cadherin (14)
, we found in these cells reduction and disruption of the CAR signal at sites of cell-cell contact after Raf-1 activation in the absence of changes in overall protein levels (Fig. 4A
, and data not shown). These findings suggest that Raf-1 expression might be sufficient to disrupt CAR expression at the cell surface. As a potential mechanism, protein redistribution from the cell surface into the cytoplasm (e.g., into lysosomes) could play a role. At present, we are conducting experiments addressing these possibilities.
Reexpression of CAR at the plasma membrane after MEK inhibition was sufficient to increase the uptake of adenovirus. This effect seems to depend on CAR specifically, because preincubation with recombinant adenovirus fiber protein or CAR-Fc fusion protein blocked virus uptake after treatment with the MEK inhibitors. We were also able to demonstrate that killing of cells treated with MEK inhibitors by wild-type adenovirus and the mutant adenovirus ONYX-015, which replicates selectively in cells with loss of a functional p53 pathway (2 , 17) , was significantly enhanced. In addition, we observed an increased virus burst from treated cells, indicating the possibility that more efficient viral entry into cells enhanced viral replication and spread. It is noteworthy that activation of ERK after adenovirus entry has been reported (29) and that for replication of coxsackievirus, activation of ERK is actually necessary (30 , 31) . It is therefore conceivable that ERK activity is also required for adenovirus replication. Thus, pharmacological inhibition of MEK might impact on viral cytotoxicity differentially, depending on the timing of inhibitor treatment.
Recently, increased CAR expression and enhanced adenovirus infectivity were also found in cancer cells after treatment with the histone deacetylase inhibitor FR901228 (32) . Because the mechanism of action of such compounds impacts on the transcriptional activity of an unknown number of genes, it is unclear at present which molecular events are contributing to this effect. Nevertheless, it is noteworthy that direct activation of histone deacetylase-4 by constitutively active MEK has been reported (33) , suggesting the possibility that treatment with histone deacetylase inhibitors abolishes Raf-MEK-ERK mediated effects.
Our results could be of direct relevance for the development of adenovirus-based treatments of cancer because inhibitors of Raf-1-MEK signaling are already being studied in clinical Phase I-II studies (34) . We envision that combined treatment of tumors in which the Raf-MEK-ERK pathway is activated with MEK inhibitors, or agents that oppose MEK activity indirectly, and adenovirus-based cancer therapeutics could have synergistic antitumor effects.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to M. A.), an American Cancer Society Individual Research Award, a grant from the UCSF Research Evaluation and Allocation Committee, the Hellman Family Award, and by funds from Onyx Pharmaceuticals, Inc. (to W. M. K.). 
2 To whom requests for reprints should be addressed, at UCSF Comprehensive Cancer Center, Box 0128, San Francisco, CA 94143. Phone: (415) 502-2844; Fax: (415) 502-3179; E-mail: korn{at}cc.ucsf.edu
3 The abbreviations used are: CAR, coxsackievirus and adenovirus receptor; ERK, extracellular signal-regulated kinase; MEK, mitogen activated protein/extracellular signal-regulated kinase kinase; MDCK, Madin-Darby canine kidney; EGFP, enhanced green-fluorescent protein; FACS, fluorescence-activated cell sorting; PI3K, phosphatidylinositol-3-kinase; 4-HT, 4-hydroxytamoxifen; 6FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; ß-GUS, ß-glucuronidase; FBS, fetal bovine serum; MOI, multiplicity of infection; CPE, cytopathic effect; DAPI, 4',6-diamidino-2-phenylindole.
4 W. M. Korn et al., Expression of receptors for adenovirus in colorectal cancer metastases, manuscript in preparation.
Received 11/19/01. Accepted 2/27/03.
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) or DMSO (as a control; 
) by FACS analysis of cells infected with a GFP-expressing, nonreplicating adenovirus (Ad-GFP) at a MOI of 10. B, HCT116 cells treated with DMSO (as a control; 