AJP - GI Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 292: G182-G190, 2007. First published August 17, 2006; doi:10.1152/ajpgi.00201.2006
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/G182    most recent
00201.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Taglia, L.
Right arrow Articles by Benya, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Taglia, L.
Right arrow Articles by Benya, R. V.

HORMONES AND SIGNALING

Gastrin-releasing peptide mediates its morphogenic properties in human colon cancer by upregulating intracellular adhesion protein-1 (ICAM-1) via focal adhesion kinase

Lauren Taglia, Damien Matusiak, Kristina A. Matkowskyj, and Richard V. Benya

Department of Medicine, University of Illinois at Chicago and Chicago Veterans Administration Medical Center (West Side Division), Chicago, Illinois

Submitted 10 May 2006 ; accepted in final form 8 August 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Gastrin-releasing peptide (GRP) and its receptor (GRPR) act as morphogens when expressed in colorectal cancer (CRC), promoting the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling and retarding tumor cell metastasis by enhancing cell-matrix attachment. Although we have shown that these processes are mediated by focal adhesion kinase (FAK), the downstream target(s) of GRP-induced FAK activation are not known. Since osteoblast differentiation is mediated by FAK-initiated upregulation of ICAM-1 (Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. J Biol Chem 278: 45368–45374, 2003), we determined whether GRP-induced activation of FAK alters ICAM-1 expression in CRC and, if so, determined the contribution of ICAM-1 to mediating GRP's morphogenic properties. Caco-2 and HT-29 cells variably express GRP/GRPR. These cells only express ICAM-1 when GRPR are present. In human CRC, GRPR and ICAM-1 are only expressed by better differentiated tumor cells, with ICAM-1 located at the basolateral membrane. ICAM-1 expression was only observed subsequent to GRPR signaling via FAK. To study the effect of ICAM-1 expression on tumor cell motility, CRC cells expressing GRP, GRPR, and ICAM-1 were cultured in the presence and absence of GRPR antagonist or monoclonal antibody to ICAM-1. CRC cells engaged in directed motility in the context of remodeling and were highly adherent to the extracellular matrix, only in the absence of antagonist or ICAM-1 antibody. These data indicate that GRP upregulation of ICAM-1 via FAK promotes tumor cell motility and attachment to the extracellular matrix.

bombesin; morphogen; motogen

GASTRIN-RELEASING PEPTIDE (GRP) is a 27-amino acid protein (14) that is the mammalian homolog of bombesin, originally isolated from the skin of the frog Bombina bombina (15). Bombesin and GRP are pharmacologically indistinguishable, possess identical potencies and abilities to activate both small and large G proteins, and are used interchangeably (4, 14, 29). GRP binds to a specific 7-transmembrane-spanning G protein-coupled (heptaspanning) receptor (GRPR), with the mouse and human receptors having been cloned and sequenced (3, 10, 53). Stimulation of the GRPR regulates the normal function of many tissues, including those in the central nervous (1, 6, 40), immune (12), and pulmonary systems (54). However, the most important property of bombesin/GRP may well be its ability to act as a morphogen in colorectal cancer (CRC).

Although epithelial cells lining the adult gastrointestinal tract do not normally express GRP/GRPR, when aberrantly expressed in CRC these proteins promote the assumption of a better differentiated phenotype by regulating cell motility in the context of remodeling (7, 8) and retard tumor cell metastasis by enhancing cell attachment to the extracellular matrix (19). Our recent investigations suggested that GRP/GRPR mediate their morphogenic properties via focal adhesion kinase (FAK) phosphorylation (19, 36). Yet since FAK can act as a signaling intermediary, this observation did not fully explain how FAK activation in and of itself might mediate GRP/GRPR's morphogenic properties.

A recent study of osteoblast-to-osteoclast differentiation indicated that this process critically included FAK-mediated upregulation of intracellular adhesion molecule-1 (ICAM-1) (42). Although this study examined FAK-initiated ICAM-1 upregulation in the context of extracellular matrix-integrin interactions, FAK has been shown to upregulate ICAM-1 independently of integrins in other cells, such as those within the synovium, by acting as a signaling intermediary (28, 43). Hence the purpose of this study was to determine whether GRP/GRPR upregulates ICAM-1 via FAK and, if so, to determine the contribution of this protein to mediating GRP/GRPR's morphogenic properties.

In this study we show that ICAM-1 mediates GRP's promotion of tumor cell motility in the context of remodeling and attachment to the extracellular matrix. This is particularly important since, although it is known that ICAM-1 can be aberrantly expressed in CRC and suppress cancer progression, the mechanism has been believed to be due to this molecule's activation of the host immune surveillance system (2, 13, 16, 25, 26, 58, 62). In contrast, we herein demonstrate that ICAM-1 mediates many if not all of GRP/GRPR's morphogenic effects, providing evidence for a hitherto unappreciated mechanism whereby ICAM-1 expression might promote CRC differentiation and retard metastasis. Thus these data not only identify the mechanism of GRP/GRPR action in CRC but also support adding ICAM-1 to the small but growing list of CRC morphogens.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Anhydrous ethanol (100%, 95%) and xylene were purchased from Pharmco Products (Brookfield, CT). Wash buffer, target retrieval solution, protein block serum, antibody diluent, LSAB2 system-horseradish peroxidase (HRP), liquid DAB substrate-chromogen system, EnVision+ HRP (DAB)-rabbit system, and automated hematoxylin were all from DAKO (Carpenteria, CA). Actin, phosphorylated tyrosine 397 (p-Y397) of FAK and ICAM-1 antibodies, as well as goat anti-rabbit IgG-HRP, were from Santa Cruz Biotechnologies (Santa Cruz, CA). Alexa 488 and Alexa 568 conjugated IgG were purchased from Molecular Probes (Eugene, OR). Auto/Iodine, Redusol, and PVDF membranes were from Fisher Scientific (Pittsburgh, PA). Transwell permeable supports were from Corning (Acton, MA). Mammalian protease inhibitor cocktail was purchased from Sigma (St. Louis, MO). ECL plus Western blotting detection system was from Amersham (Piscataway, NJ). The 30% acrylamide/bis solution and Precision Plus protein standards were from Bio-Rad Laboratories (Richmond, CA). BCA protein assay kit was from Pierce (Rockford, IL).

As previously described (7), GRP expression was detected by use of a polyclonal rabbit anti-human peptide antibody directed to amino acids 14 to 27 from Abcam, whereas GRPR expression was detected by using a polyclonal rabbit anti-peptide antibody directed to the distal third intracellular loop of the mouse and human GRP-R (CVEGNIHVKKQIESRKR). The GRPR-specific antagonist D-Phe6(bombesin) methyl ester was kindly provided by Dr. David Coy (Tulane University, New Orleans, LA).

Tumor selection and assessment of differentiation. Human CRC were randomly selected from the Chicago Veterans Administration Medical Center Gastrointestinal Tissue Bank. This resource contains paraffin blocks of all CRC removed at this institution since its opening in 1956. For this study, 10 CRC resected between 1995 and 2005 were randomly selected; all tissues studied were different from, and have not been previously evaluated as a part of, prior evaluations performed by our group (7, 21, 23, 34, 36, 38, 39). The 10 CRC contained 61 separate regions of distinct differentiation, approximately equally distributed between those that were well, moderately, or poorly differentiated (Table 1). For each region of distinct differentiation, between 50 and 2,000 cells were analyzed, with the mean number of cells analyzed per each region of differentiation ranging between 75 and 580 cells.


View this table:
[in this window]
[in a new window]

  		Table 1. Characterization of human CRC studied

 
Nonconfocal photomicrographs were obtained using a SPOT RT digital scanning camera from Diagnostic Instruments (Sterling Heights, MI) attached to a Nikon E600 microscope system. Tumor differentiation was performed using a three-grade classification system as previously described (8). Well-differentiated tumors were defined by the presence of well-formed glands containing malignant columnar cells displaying small regular nuclei. The complete absence of gland formation, or the presence of bizarrely shaped glands, identified poorly differentiated tumors. Moderately differentiated tumors possessed well-formed glands, but the cells were less columnar or frankly cuboidal, with reduced cell polarity and more dysplastic nuclei than those observed in well-differentiated tumors.

Immunohistochemistry: confocal microscopy. Cells were seeded on collagen-coated Transwell inserts at various densities for 18 h. Cells were then fixed with 3.7% formaldehyde in PBS, washed with PBS, and permeabilized for 2 min using Triton X-100. Cells were then blocked with 5% normal goat serum for 30 min. Membranes were incubated with primary antibody overnight at 4°C in a humidity chamber. Following PBS washing, membranes were incubated with the either GRP (1:20), GRPR (1:500), ICAM-1 (1:100), actin (1:100), E-cadherin (1:100), or pY397 of FAK (1:100), followed by Alexa 568-conjugated goat anti-rabbit IgG and Alexa 488 goat anti-mouse IgG for 60 min.

For resected human CRCs a similar protocol was followed. Briefly, 4-µm-thick tissue sections were rehydrated using graded alcohols. Sections were incubated with Triton X-100 for 2 min, incubated the indicated antibody (at the same concentrations as used for cultured cells) overnight at 4°C in a humidity chamber, and later probed with conjugated secondary antibody.

In all instances, images were obtained using a Zeiss LSM 510 confocal microscope (New York, NY). Images were obtained at 1-µm-thick increments using x40 or x63 plan neoflux objectives.

Immunohistochemistry: immunoperoxidase. A modified indirect immunoperoxidase technique was performed when DAB was used in avidin-biotin complex immunohistochemistry and was performed to permit quantification (described under Quantitative immunohistochemistry). Cells were seeded on uncoated, multiwell Permanox slides in complete medium. To achieve confluence by day 4 postplating, 400,000 cells were plated per 4.5-cm2 well of a two-well slide. Cells were serum starved for 24 h before immunohistochemistry. Cells were fixed in situ in 3.7% formaldehyde in PBS for 30 min at 37°C. Slides were processed using the Envision Plus system (DAKO). Briefly, fixing solution was removed and cells were washed with TBS supplemented with 0.05% Tween 20. Endogenous peroxidase activity was blocked by using 0.03% hydrogen peroxide for 5 min at room temperature followed by washing the cells and then incubating with primary antibody for 1 h at room temperature. Cells were probed with a polymer-labeled anti-rabbit secondary antibody for 30 min, followed by DAB+ for 3 min. Slides were counterstained with Gill's hematoxylin for 2 min.

Quantitative immunohistochemistry. Chromogen abundance was quantified by determining the cumulative signal strength of the digital image file of the relevant region or cell of interest (32–34, 37). For all specimens, control tissues were processed identically and at the same time except that primary antibody was not applied. Control tissues were within 4 µm of the tested tissue and treated with secondary antibody, DAB+ and counterstained with hematoxylin for precisely the same time as the experimental tissue. Thus all differences between the experimental tissue and the control tissue are ultimately due to DAB identification of the relevant protein. The amount of chromogen per pixel was determined by subtracting the mathematical energy (EM) of the control slide (i.e., not exposed to primary antibody) from that in the homologous region of the experimental slide (i.e., exposed to primary antibody). Chromogen quantity (EM) is expressed as energy units per pixel (eu/pix). Maximal and half-maximal EM values were determined after curve fitting the data by least-squares nonlinear regression.

Cell lines and cell culture. To study the effects of FAK, we used specially modified 293 cells that, as we have previously described, expressed the dominant negative regulator for FAK-related nonkinase (FRNK) (19). Briefly, these cells were modified to express FRNK under control of a doxycycline-inducible promoter (19). Although these cells do not express FRNK under basal conditions, FRNK is rapidly upregulated after exposure to 2 µg/ml doxycycline (Clonetech) for at least 18 h (19). Cells were cultured in Opti-MEM containing HEPES buffer, sodium bicarbonate, and L-glutamine supplemented with 10% tetracycline-free fetal bovine serum, 600 mg/ml G-418, and 200 mg/ml hygromycin B; 293 cells were maintained at 37°C in a 5% CO2 atmosphere.

In contrast, Caco-2 and HT-29 cells were cultured in D-MEM/F12 containing glucose and L-glutamine supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in a 5% CO2 atmosphere in all instances.

Western analysis. Cells were grown to at least 60% confluence and then incubated in serum-free medium for 24 h. Thereafter, cell monolayers were rinsed in PBS and lysed in ice-cold RIPA (50 mM HEPES, pH 7.4; 150 mM NaCl; 1% Triton X-10; 0.1% SDS; 0.5% sodium deoxycholate; 1 mM sodium orthovanadate; 5 mM EDTA; 5 mM sodium fluoride) containing a 1:20 dilution of mammalian protease inhibitor cocktail (Sigma). In all instances, 200 µg of total cell protein were loaded into a preparative well and electrophoresed across a 10% polyacrylamide gel under denaturing and reducing conditions. Membranes were incubated with primary antibody for 2 h at the following concentrations: actin 1:100, GRPR 1:500, Y397 1:100, and ICAM-1 1:500. This was followed by two 10-min washes with TBST with immunoreactive bands visualized using a HRP-conjugated goat anti-rabbit IgG and the ECL Plus detection system.

Cell-matrix attachment. Cell adhesion to the extracellular matrix was determined as previously described (30, 56, 59). Briefly, cells were plated at a density of 50,000 cells per well in a 12-well plate. Cells were cultured for 24 h before being washed with PBS and treated for 3 h in serum-free medium alone, or in the presence of 1 µM D-Phe6 bombesin (methyl ester), ICAM-1 monoclonal antibody at a concentration of 1:50, or goat anti-mouse antibody at a concentration of 1:100 (used as a control). Cells were then exposed to 500 µl of 0.25% trypsin/1 mM EDTA for 2 min at the indicated time points, and total number of detached cells was quantified by use of a Beckman-Coulter Z2 counter (Miami, FL).

Motility. Cells were plated and cultured until 2 days postconfluent, washed, and then cultured in serum-free medium for an additional 18 h. A linear gap in the cell monolayer was generated by use of a sterile scalpel blade. Cells were then mounted in a custom-designed Leica inverted tissue culture microscope such that cells were consistently maintained at 37°C. Cell images were acquired every 5 min with a Hamamatsu 1.3 mega-pixel digital camera (Hamamatsu City, Japan). Evaluation of cell behavior was performed using Open Lab (Improvision, Coventry, UK).

Cell motility was quantified as the percentage of the original wound size, or gap, remaining over time. Three separate values were obtained for each image, and each experiment was repeated a minimum of three separate times. Images were converted into QuickTime files at native size (640 x 480) using the "Video" codec, edited using Final Cut Pro 4.0 (Apple), and exported for viewing using the "JPEG A" codec at reduced size (320 x 240) at 10 frames/s. The resultant videos can be viewed at: http://www.uic.edu/com/dom/gastro/labvideos.html.

Statistical analysis. All evaluations were performed using Excel (Microsoft, Redmond, WA), with data reported as means ± SE. Data were evaluated by paired or unpaired t-test or by ANOVA, as appropriate, using built-in statistical functions provided by Excel.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
GRP/GRPR-ICAM-1 expression and location in human CRC. We previously showed that Caco-2 and HT-29 cells variably express GRP/GRPR as a function of confluence (20). We determined the location of GRPR and ICAM-1 expression in preconfluent Caco-2 (Fig. 1) and HT-29 [data not shown (NS)] cells by confocal microscopy. GRPR were expressed primarily if not exclusively in the apical membrane of these cells (Fig. 1A). In contrast, ICAM-1 was present diffusely in the cytoplasm of Caco2 (Fig. 1B) and HT-29 cells, as well as showed evidence of being coexpressed with GRPR at the level of the apical membrane (Fig. 1C). To evaluate for expression at the level of the basolateral membrane (BLM), we used a specific antibody for FAK. Confocal images confirmed that ICAM-1 was also present in the region of the BLM (Fig. 1D). Lastly we used an antibody for actin to demonstrate that ICAM-1 was not expressed in regions mediating cell-cell contact (Fig. 1E).


Figure 1
View larger version (67K):
[in this window]
[in a new window]

  		Fig. 1. ICAM-1 location in Caco-2 cells. A–D: Z-axis planes for gastrin-releasing peptide (GRP; A, green), GRP receptor (GRPR; B, green), and ICAM-1 (C, red), with GRPR-ICAM-1 colocalization apparent at the apical membrane (D, yellow). Likewise, ICAM-1 (red) and focal adhesion kinase (FAK) (green) (E) colocalize (yellow) at the basolateral membrane. F: Caco-2 monolayer stained for actin (green) and ICAM-1 (red), showing that the latter protein is not present in regions involved in cell-cell contact.

 
We next determined whether ICAM-1 expression was likewise associated with the amount of GRP/GRPR present in resected human CRC. To do this we evaluated the expression of GRP/GRPR and ICAM-1 in 10 human CRC randomly selected from our Gastrointestinal Tissue Bank (7). In all instances, consecutive 4-µm-thick sections were evaluated for GRP, GRPR, or ICAM-1 expression, allowing for expression to be quantified [by quantitative immunohistochemistry (Q-IHC) (32–34, 37)] in the same cells within any particular cancer. Similar to what we have previously shown (7), GRP/GRPR were not expressed by nonmalignant epithelial cells lining the colon (data NS). Similarly, no evidence of ICAM-1 expression could be detected in nonmalignant epithelial cells lining the colon (Fig. 2, A and B). In contrast, and similar to what we have previously reported (7), GRPR expression was a function of tumor cell differentiation, with over 270 ± 7 eu/pix detected in well-differentiated CRC cells and 159 ± 11 eu/pix in moderately differentiated cells, but only 35 ± 3 eu/pix in poorly differentiated cells (Fig. 3). Likewise, ICAM-1 expression in CRC was also a function of CRC cell differentiation, such that high levels of expression were noted in well differentiated tumor cells (237 ± 7 eu/pix; Fig. 2, C and D; Fig. 3) and low levels in poorly differentiated cells (39 ± 6 eu/pix; Fig. 2, E and F; Fig. 3). Overall, ICAM-1 expression tightly correlated with the coexpression of GRP/GRPR in any particular cell (Fig. 3; r2 = 0.91). Importantly, and in contrast to what was observed for Caco-2 and HT-29 cells, most ICAM-1 expression (when present) was evident at the level of the BLM (Fig. 2D).


Figure 2
View larger version (103K):
[in this window]
[in a new window]

  		Fig. 2. Expression of ICAM-1 in normal colonic epithelium and in human colon cancer as a function of tumor cell differentiation. A, C, and E: differential interference contrast (DIC) images of normal adult colon (A) as well as well-differentiated (C) and a poorly differentiated (E) tumors. B,D,F: confocal images of normal colonic epithelium (B) stained for actin (green) and ICAM-1 (red) as well as well-differentiated (D) and poorly differentiated (F) tumor cells. Boxes in C and E indicate region of DIC image magnified in the adjacent confocal image. White arrow in D identifies the margin of the basement membrane.

 

Figure 3
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 3. Quantification of GRP/GRPR and ICAM-1 coexpression in human colorectal cancer (CRC). Randomly selected human CRC were obtained from the Gastrointestinal Tumor Database and processed immunohistochemically for GRP, GRPR, and ICAM-1 expression, and protein amount in the same cells was quantified as described in METHODS. Data represent all individual cells evaluated and represent the evaluations of 10 separate human CRC as a function of tumor cell differentiation. Error bars for individual data points not apparent due to their small size, with data expressed as energy units per pixel (eu/pix).

 
We next investigated the kinetics of ICAM-1 expression (Fig. 4). Cells were plated to confluence, washed, cultured in serum-free medium for 24 h, and then wounded with a sterile razor blade. At the indicated time point, protein expression was quantified by DAB-based immunohistochemistry as previously described (32–34, 37). GRPR were rapidly upregulated postwounding, with maximal expression noted at time (t) = 6.8 ± 1.3 h and half-maximal expression at 2.8 ± 1.6 h. In contrast, ICAM-1 expression lagged slightly, albeit significantly, such that maximal expression was noted at t = 7.0 ± 1.7 h and half-maximal expression at 4.0 ± 0.6 h (P < 0.001 for both conditions, paired t-test; Fig. 4). Similar results were obtained for HT-29 cells evaluated similarly (data NS). Finally, cells were coincubated with a GRPR antagonist D-Phe6(bombesin) methyl ester (11, 61), and ICAM-1 expression was assessed postwounding. Incubating wounded cells with this antagonist completely eliminated ICAM-1 expression (Fig. 4).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 4. Kinetics of GRPR (bullet) and ICAM-1 expression in the absence ({square}) or presence of antagonist D-Phe6(bombesin) methyl ester ({blacktriangleup}) in Caco-2 cells during the first 10 h postwounding, as determined by quantitative immunohistochemistry. Data represent means ± SE for a minimum of 3 separate experiments, with data expressed as eu/pix. Because of the very small SE, error bars are not visible for all data points.

 
Role of FAK in GRPR-initiated ICAM-1 expression. We previously showed that GRPR-induced phosphorylation of FAK at Y397 (19, 36) critically mediated many of this protein's morphogenic properties. Because FAK is known to upregulate ICAM-1 (28, 42, 43), whereas GRP is known to upregulate FAK, we determined whether activated GRPR upregulated ICAM-1 expression in Caco-2 (data NS) and HT-29 cells (Fig. 5). Preconfluent cells were cultured in serum-free medium for 24 h in the absence (Fig. 5, left) or presence (Fig. 5, right) of the GRPR-specific antagonist D-Phe6(bombesin) methyl ester. Preconfluent GRP-secreting, GRPR-expressing cells showed evidence of FAK activation by virtue of tyrosine 397 being phosphorylated, consistent with what we have previously published, and ICAM-1 being expressed. In contrast, abrogating GRPR signaling using D-Phe6(bombesin) methyl ester eliminated tyrosine 397 phosphorylation as well as ICAM-1 expression.


Figure 5
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 5. ICAM-1 expression and FAK tyrosine (Y) 397 phosphorylation in HT-29 cells requires GRPR signaling. Preconfluent HT-29 cells (left) were cultured in serum-free medium for 24 h, and the proteins expressed were determined by using a Mini-Protean II Multi-Screen, allowing for identical amounts of protein to be probed simultaneously with different antibodies. Preconfluent HT-29 cells treated similarly except for exposure to the GRPR antagonist D-Phe6(bombesin) methyl ester (ME) for 24 h (right) failed to show evidence of ICAM-1 expression or Y397 phosphorylation. Gel representative of 3 independently performed experiments.

 
To confirm the role of FAK in regulating ICAM-1 expression, we studied GRP/GRPR-expressing 293 cells that had been modified to express FRNK, the dominant negative inhibitor of FAK, under control of a doxycycline-sensitive inducible promoter (19). Under basal conditions, GRP autocrine activation of its cognate receptor results in Y397 phosphorylation of FAK and ICAM-1 expression (Fig. 6, left). When the same cells are now exposed to doxycycline, thereby activating FRNK, no change in GRPR expression is noted, but both FAK phosphorylation at Y397 and ICAM-1 expression are completely ablated (Fig. 6, right). Thus GRP-induced upregulation of ICAM-1 occurs via FAK.


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. Regulation of GRPR-dependent ICAM-1 expression via FAK. GRP/GRPR-expressing HEK-293 cells were modified to express FAK-related nonkinase (FRNK), the dominant-negative inhibitor of FAK, under control of a doxycycline-sensitive promoter, and treated as indicated. In the absence of doxycycline (left), FAK is phosphorylated at Y397 and ICAM-1 is present. In the presence of doxycycline (right), there is no change in GRPR expression whereas FAK phosphorylation at Y397 is completely abrogated and ICAM-1 expression is absent. Western blotting analysis was performed using a Mini-Protean II Multi-Screen, allowing for identical amounts of protein to be probed simultaneously with different antibodies, with actin shown to indicate identical amounts of protein were evaluated in both conditions. The gel is representative of 3 independently performed experiments.

 
Contribution of ICAM-1 to mediating cell-ECM attachment. We have previously shown that GRPR activation enhances cell attachment to the extracellular matrix, thereby retarding metastasis, and increases cell motility in the context of remodeling, promoting the assumption of a better differentiated phenotype (19). To assess cell attachment, we plated Caco-2 cells at subconfluent densities for 24 h to promote GRP/GRPR expression. Cells were then washed in serum-free PBS in the absence or presence of exogenous GRP, the antagonist D-Phe6(bombesin) methyl ester, control antibody, or monoclonal antibody directed to ICAM-1 for 3 h. Cells were then washed again followed by exposure to 0.25% trypsin for the indicated time, and detached cells were counted. When exposed to trypsin in serum-free medium for 10 min all cells were detached, whereas exposure for 1 min resulted in ~50% of attached cells detaching. We therefore used 1 min exposure to trypsin as our baseline condition so that alterations in avidity of cell attachment in either direction could be assessed.

Exposure to exogenous GRP (1 µM) for 3 h markedly increased Caco-2 cell attachment to the ECM by 62.3 ± 2.1% (P < 0.0001, paired t-test) (Fig. 7). In contrast, exposure to the GRPR-specific antagonist D-Phe6(bombesin) methyl ester decreased Caco-2 cell attachment by 53.7 ± 3.9% compared with cells exposed to medium and autocrine GRP alone (P < 0.0001, paired t-test). Although ICAM-1 antagonists do not exist, we explored whether the commercially available antibody used in our immunohistochemical studies could act to inhibit ICAM-1 action, similar in concept to what we have previously done to inhibit the action of the galanin-1 receptor (35). Thus we exposed cells to monoclonal antibody directed to ICAM-1 for 3 h. In so doing we noted a similar 48.9 ± 3.3% decrease in cell attachment to the ECM compared with cells exposed to serum-free medium alone (P < 0.0001, paired t-test) (Fig. 7). In contrast, rabbit anti-human IgG antibody (1:100) used as a control had no impact the avidity of Caco-2 cell attachment to the ECM (data NS).


Figure 7
View larger version (28K):
[in this window]
[in a new window]

  		Fig. 7. Effect of GRPR and ICAM-1 expression in Caco-2 cells on cell attachment. Cells were treated with 0.25% trypsin/EDTA for 10 min or 1 min. Treatment with 1 µM exogenous GRP promoted cell attachment whereas treatment with 1 µM GRPR antagonist D-Phe-6(bombesin) methyl ester or ICAM-1 antibody decreased cell attachment. Data represent means ± SE for a minimum of 3 separate experiments, with each experiment performed in duplicate, and horizontal lines indicating significantly different results between identified conditions. *Bars are significantly different (P < 0.05).

 
Contribution of ICAM-1 to mediating cell motility in the context of remodeling. We next evaluated motility in the context of remodeling by culturing Caco-2 cells to confluence, wounding them with a sterile razor blade, and tracking cell position at the indicated time points. Similar to what we have previously described (20), Caco-2 cells migrated rapidly into the gap created by the razor blade, and once the gap was closed no further cell movement could be detected, consistent with the motility occurring to retain tissue architecture and not representative of greater malignancy (Fig. 8) (see video at http://www.uic.edu/com/dom/gastro/Caco2.videos.html). When cultured in the presence of the antagonist D-Phe6(bombesin) methyl ester, and similar to what we have previously reported (20), cells initially migrated into the gap, consistent with the fact that GRPR are not originally present, with migration attenuating from 3 h onward, consistent with increasing receptor expression (Fig. 8). Lastly, we used ICAM-1 antibody to probe the contribution of this protein to mediating GRP-initiated cell motility. Whereas rabbit anti-human IgG antibody (1:100) used as a control had no impact on cell motility (Fig. 8), ICAM-1 antibody used at the same concentration attenuated Caco-2 cell motility similarly as GRPR antagonist D-Phe6(bombesin) methyl ester (Fig. 8) (see videos of both conditions at http://www.uic.edu/com/dom/gastro/labvideos.html). Specifically, ICAM-1 antibody had no effect on attenuating cell migration into the gap immediately after wounding, consistent with there being no ICAM-1 expressed. After the first 3 h, however, there was progressive attenuation in Caco-2 cell motility, with the attenuation paralleling the increase in ICAM-1 expression (Fig. 4). Thus these data strongly suggest that ICAM-1 mediates GRP's morphogenic actions of enhancing cell attachment and promoting directed cell motility.


Figure 8
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 8. Caco-2 cell motility in the context of wound remodeling was determined by quantifying the amount of gap or wound between cell edges and expressed as the % of wound remaining. Whereas untreated cells ({blacklozenge}) demonstrate rapid motility into the wound, migration was significantly attenuated in the presence of antagonist [D-Phe6(bombesin) methyl ester ({circ})]. Similarly, cultured wounded Caco-2 cells in the presence of ICAM-1 antibody ({blacksquare}) attenuated cell motility whereas control anti-human IgG ({blacktriangleup}) showed no effect. Data represent means ± SE for a minimum of 3 separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Epithelial cells lining the adult colon do not normally express ICAM-1 but can be expressed subsequent to malignant transformation (13, 26, 57). When present, significant evidence exists showing that ICAM-1 expression decreases CRC metastasis. Lymph node or liver metastases are decreased in patients whose primary tumor expresses ICAM-1 compared with those whose tumor does not (31), whereas lower numbers of ICAM-1 positive cells are observed in primary tumors that metastasize (62). Direct evidence for ICAM-1 expression decreasing CRC metastasis comes from a recent study that, using a nude mouse model, noted decreased liver metastases in the human CRC cell line LM-H3 ectopically expressing ICAM-1 compared with cells not expressing this protein (55). However, the regulation of ICAM-1 expression in CRC is not known, whereas the mechanism whereby ICAM-1 is believed to decrease metastasis is less than convincing.

ICAM-1 is believed to decrease tumor cell metastasis by promoting heterotypic cell-cell contact with inflammatory cells (2, 13, 16, 25, 26, 58, 62). Since ICAM-1 expression in CRC correlates with the number of inflammatory cells within a particular tumor (26), and given that ICAM-1 promotes the attachment of such cells to human CRC cell lines (26), this protein has been proposed to play a role in promoting lymphocyte-mediated tumor killing (26, 58). Indeed, even the study whereby ICAM-1 was ectopically expressed in LM-H3 cells thereby decreasing metastasis suggested that this occurred as a result of enhanced binding of peripheral blood mononuclear cells to the tumor cells and subsequent tumor cell lysis (55). Yet the data presented herein suggest an alternative mechanism, namely that ICAM-1 enhances tumor cell attachment to the extracellular matrix. Along with contributing to tumor cell architecture by promoting motility in the context of remodeling, ICAM-1 appears to be acting as a morphogen, mediating the actions of GRP upon autocrine activation of its cognate receptor.

GRP and its receptor are typically studied in the context of their ability to act as mitogens when aberrantly expressed in cancer, including those derived from the colon (17, 18, 44, 49, 50). Yet we have shown that, when aberrantly expressed in CRC, GRP's mitogenic properties are subordinate to its morphogenic properties (reviewed in Ref. 24). Like ICAM-1, GRP/GRPR are not expressed by epithelial cells lining the normal adult human colon, but when aberrantly expressed in CRC they are present in better differentiated cancer cells but not poorly differentiated cells or metastases (7). Our study of GRPR–/– mice provided direct evidence for GRP/GRPR acting as morphogens and regulating tumor cell differentiation. Whereas GRPR–/– mice develop CRC that become progressively more poorly differentiated, those developing in wild-type GRPR-expressing mice are well differentiated (8). Since the differentiation of solid tumors correlates with patient survival for all tumors studied (5, 9, 27, 41, 45–47, 51, 52, 60), including those developing in the colon (22, 63), morphogens not only regulate tumor appearance but also regulate behaviors altering the propensity of a tumor cell to metastasize.

We previously identified biophysical properties that are surrogate markers for tumor cell differentiation and metastasis, including tumor cell migration in the context of remodeling and cell attachment to the extracellular matrix (19). Although FAK phosphorylation has been shown to mediate GRP-stimulated cell proliferation (48), we have also shown that FAK phosphorylation at tyrosine 397 regulates tumor cell motility in the context of remodeling and attachment to the extracellular matrix (19, 36). However, since FAK can serve as a signaling molecule, these observations begged the question as to what downstream proteins might exist mediating GRP's morphogenic effects. The recent studies showing that osteoblast to osteoclast differentiation critically involved ICAM-1 upregulation via a FAK-dependent pathway, along with our prior studies indicating that GRP/GRPR regulate CRC differentiation via FAK, suggested that this downstream protein might be ICAM-1.

In this paper we show for the first time that autocrine activation of the GRPR causes ICAM-1 to be rapidly expressed via a FAK-dependent signaling cascade. We also show that ICAM-1 is primarily expressed at the level of the BLM, where it mediates GRP's morphogenic properties. We specifically show that ICAM-1 mediates tumor cell attachment to the extracellular matrix. In so doing ICAM-1 would be expected to prevent cells from detaching from the primary tumor mass and thus attenuate or eliminate metastasis. Thus this represents a process that is completely independent of this protein's previous documented ability to improve patient survival by promoting the binding of inflammatory cells to CRCs (2, 13, 16, 25, 26, 58, 62).

Equally important, we also show for the first time that ICAM-1, by mediating tumor cell motility in the context of remodeling, acts as a motogen. The qualification that this motility occurs only in the context of remodeling is important. Cells of greater malignancy show no evidence of contact inhibition and would in our wounding assay show evidence of intercalating between one another after the gap was filled. In contrast, we herein demonstrate that after the wound is filled cell motility ceases. Thus this directed motility is similar in principle to what is observed with nonmalignant cells as they maintain the architecture characteristic of the organ system whence they reside or is required as a part of organogenesis. Hence our bioassay quantifying directed cell motility measures a core property of morphogens in malignancy. As such, our data not only identify the mechanism of GRP/GRPR action as a morphogen when aberrantly expressed in CRC but also indicate that ICAM-1 itself is a morphogen in at least this type of tumor.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Cancer Institute Grant CA-094346 and a Veterans Affairs Merit Review award (to R. V. Benya).


   ACKNOWLEDGMENTS
 
The authors thank Dr. David Coy (Tulane University, New Orleans, LA) for generous provision of GRPR antagonist D-Phe6(bombesin) methyl ester and Dr. Grace Guzman (University of Illinois at Chicago) for assistance regarding histopathological assessments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. V. Benya, Dept. of Medicine, Univ. of Illinois at Chicago, 840 South Wood St. (M/C 716), Chicago, IL 60612 (e-mail: rvbenya{at}uic.edu)

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


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Albers HE, Liou SY, Stopa EG, Zoeller RT. Interaction of colocalized neuropeptides: functional significance in the circadian timing system. J Neurosci 11: 846–851, 1991.[Abstract]
  2. Alexiou D, Karayiannakis AJ, Syrigos KN, Zbar A, Kremmyda A, Bramis I, Tsigris C. Serum levels of E-selectin, ICAM-1 and VCAM-1 in colorectal cancer patients: correlations with clinicopathological features, patient survival and tumour surgery. Eur J Cancer 37: 2392–2397, 2001.[CrossRef][ISI][Medline]
  3. Battey JF, Way JM, Corjay MH, Shapira H, Kusano K, Harkins R, Wu JM, Slattery T, Mann E, Feldman RI. Molecular cloning of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells. Proc Natl Acad Sci USA 88: 395–399, 1991.[Abstract/Free Full Text]
  4. Benya RV, Kusui T, Pradhan TK, Battey JF, Jensen RT. Expression and characterization of cloned human bombesin receptors. Mol Pharmacol 47: 10–20, 1995.[Abstract]
  5. Birchmeier W, Behrens J, Weidner KM, Hulsken J, Birchmeier C. Epithelial differentiation and the control of metastasis in carcinomas. Curr Top Microbiol Immunol 213: 117–135, 1996.
  6. Brown MR, Carver K, Fisher LA. Bombesin: central nervous system actions to affect the autonomic nervous system. Ann NY Acad Sci 547: 174–182, 1988.[ISI][Medline]
  7. Carroll RE, Matkowskyj KA, Chakrabarti S, McDonald TJ, Benya RV. Aberrant expression of gastrin-releasing peptide and its receptor by well differentiated colon cancers in humans. Am J Physiol Gastrointest Liver Physiol 276: G655–G665, 1999.[Abstract/Free Full Text]
  8. Carroll RE, Matkowskyj KA, Tretiakova MS, Battey JF, Benya RV. Gastrin-releasing peptide is a mitogen and morphogen in murine colon cancer. Cell Growth Differ 11: 385–393, 2000.[Abstract/Free Full Text]
  9. Coindre JM. Histologic grading of adult soft tissue sarcomas. Verh Dtsch Ges Pathol 82: 59–63, 1998.[Medline]
  10. Corgay MH, Dobrzanski DJ, Way JM, Viallet J, Shapira H, Worland P, Sausville EA, Battey JF. Two distinct bombesin receptor subtypes are expressed and functional in human lung carcinoma cells. J Biol Chem 266: 18771–18779, 1991.[Abstract/Free Full Text]
  11. Coy DH, Jensen RT, Jiang NY, Lin JT, Bogden AE, Moreau JP. Systematic development of bombesin/gastrin-releasing peptide antagonists. J Natl Cancer Inst Monogr 13: 133–139, 1992.
  12. De La Fuente M, Del Rio M, Hernanz A. Stimulation of natural killer and antibody-dependent cellular cytotoxicity activities in mouse leukocytes by bombesin, gastrin-releasing peptide and neuromedin C: involvement of cyclic AMP, inositol 1,4,5-trisphosphate and protein kinase C. Ann Oncol 4: 499–507, 1993.[Abstract/Free Full Text]
  13. Dippold W, Wittig B, Schwaeble W, Mayet W, Meyer zum Buschenfelde KH. Expression of intercellular adhesion molecule 1 (ICAM-1, CD54) in colonic epithelial cells. Gut 34: 1593–1597, 1993.[Abstract/Free Full Text]
  14. Erspamer V. Discovery, isolation and characterization of bombesin-like peptides. Ann NY Acad Sci 547: 3–9, 1988.[ISI][Medline]
  15. Erspamer V, Erspamer GF, Inslevini M, Negri L. Occurrence of bombesin and alytensin in extracts of the skin of three European discoglossid frogs and pharmacological actions of bombesin on extravascular smooth muscle. Br J Pharmacol 45: 333–348, 1972.[ISI][Medline]
  16. Forest V, Pierre F, Bassonga E, Meflah K, Olivier C, Menanteau J. Apc+/Min colonic epithelial cells express TNF receptors and ICAM-1 when they are co-cultured with large intestine intra-epithelial lymphocytes. Cell Immunol 223: 70–76, 2003.[CrossRef][ISI][Medline]
  17. Frucht H, Gazdar A, Jensen RT. Human colon cancer cell line NCI-H716 expresses functional bombesin receptors. Proc Am Assoc Cancer Res 32: 47, 1991.
  18. Frucht H, Gazdar AF, Park JA, Oie H, Jensen RT. Characterization of functional receptors for gastrointestinal hormones on human colon cancer cells. Cancer Res 52: 1114–1122, 1992.[Abstract/Free Full Text]
  19. Glover S, Delaney M, Dematte C, Kornberg L, Frasco M, Tran-Son-Tay R, Benya RV. Phosphorylation of focal adhesion kinase tyrosine 397 critically mediates gastrin-releasing peptide's morphogenic properties. J Cell Physiol 199: 77–88, 2004.[CrossRef][ISI][Medline]
  20. Glover S, Nathaniel R, Shakir L, Perrault C, Anderson R, Tran-Son-Tay R, Benya RV. Transient upregulation of GRP and its receptor critically regulates colon cancer cell motility during remodeling. Am J Physiol Gastrointest Liver Physiol 288: G1274–G1282, 2005.[Abstract/Free Full Text]
  21. Glover SC, Tretiakova MS, Carroll RE, Benya RV. Increased frequency of GRPR gene mutations during colon adenocarcinoma progression. Mol Carcinog 37: 5–15, 2003.[CrossRef][ISI][Medline]
  22. Hase K, Shatney C, Johnson D, Trollope H, Vierra M. Prognostic value of tumor "budding" in patients with colorectal cancer. Dis Colon Rectum 36: 627–635, 1993.[CrossRef][ISI][Medline]
  23. Hu Y, Benya RV, Carroll RE, Diamond AM. Allelic loss of the gene for the GPX1 selenium-containing protein is a common event in cancer. J Nutr 135: 3021S–3024S, 2005.[Abstract/Free Full Text]
  24. Jensen JG, Carroll RE, Benya RV. The case for gastrin-releasing peptide acting as a morphogen when it and its receptor are aberrantly expressed in cancer. Peptides 22: 689–699, 2001.[CrossRef][ISI][Medline]
  25. Kaiserlian D, Rigal D, Abello J, Revillard JP. Expression, function and regulation of the intercellular adhesion molecule-1 (ICAM-1) on human intestinal epithelial cell lines. Eur J Immunol 21: 2415–2421, 1991.[ISI][Medline]
  26. Kelly CP, O'Keane C, Orellana J, Schroy PC 3rd, Yang S, LaMont T, Brady HR. Human colon cancer cells express ICAM-1 in vivo and support LFA-1-dependent lymphocyte adhesion in vitro. Am J Physiol Gastrointest Liver Physiol 263: G864–G870, 1992.[Abstract/Free Full Text]
  27. Kowalski LP, Medina JE. Nodal metastases: predictive factors. Otolaryngol Clin North Am 31: 621–637, 1998.[CrossRef][ISI][Medline]
  28. Koyama Y, Tanaka Y, Saito K, Abe M, Nakatsuka K, Morimoto I, Auron PE, Eto S. Cross-linking of intercellular adhesion molecule 1 (CD54) induces AP-1 activation and IL-1beta transcription. J Immunol 157: 5097–5103, 1996.[Abstract]
  29. Kroog G, Sainz E, Worland P, Akeson M, Benya RV, Jensen RT, Battey JF. The gastrin-releasing peptide receptor is rapidly phosphorylated by a kinase other than protein kinase C after exposure to agonist. J Biol Chem 270: 8217–8224, 1995.[Abstract/Free Full Text]
  30. Lin VC, Eng AS, Hen NE, Ng EH, Chowdhury SH. Effect of progesterone on the invasive properties and tumor growth of progesterone receptor-transfected breast cancer cells MDA-MB-231. Clin Cancer Res 7: 2880–2886, 2001.[Abstract/Free Full Text]
  31. Maeda K, Kang SM, Sawada T, Nishiguchi Y, Yashiro M, Ogawa Y, Ohira M, Ishikawa T, Hirakawa YS, Chung K. Expression of intercellular adhesion molecule-1 and prognosis in colorectal cancer. Oncol Rep 9: 511–514, 2002.[ISI][Medline]
  32. Matkowskyj K, Glover S, Benya R. Quantitative immunohistochemistry: an algorithm measuring cumulative signal strength and receptor number. Microsc Anal 18: 5–6, 2004.
  33. Matkowskyj KA, Cox R, Benya RV. Quantitative immunohistochemistry by determining the norm of the image file. In: Immunohistochemistry of In Situ Hybridization of Human Carcinomas, edited by Hayat MA. Burlington, MA: Academic, 2006, p. 279–284.
  34. Matkowskyj KA, Cox R, Jensen RT, Benya RV. Quantitative immunohistochemistry by measuring cumulative signal strength accurately measures protein concentration. J Histochem Cytochem 51: 205–214, 2003.[Abstract/Free Full Text]
  35. Matkowskyj KA, Danilkovic A, Marrero JA, Savkovic S, Hecht G, Benya RV. Galanin-1 receptor up-regulation mediates the excess colonic fluid production caused by infection with enteric pathogens. Nat Med 6: 1048–1051, 2000.[CrossRef][ISI][Medline]
  36. Matkowskyj KA, Keller K, Glover S, Kornberg L, Tran-Son-Tay R, Benya RV. Expression of GRP and its receptor in well differentiated colon cancer cells correlates with the presence of focal adhesion kinase phosphorylated at tyrosines 397 and 407. J Histochem Cytochem 51: 1041–1048, 2003.[Abstract/Free Full Text]
  37. Matkowskyj KA, Schonfeld D, Benya RV. Quantitative immunohistochemistry by measuring cumulative signal strength using commercially available software Photoshop and Matlab. J Histochem Cytochem 48: 303–311, 2000.[Abstract/Free Full Text]
  38. Matusiak D, Glover S, Nathaniel R, Matkowskyj K, Yang J, Benya RV. Neuromedin B and its receptor are mitogens in both normal and malignant epithelial cells lining the colon. Am J Physiol Gastrointest Liver Physiol 288: G718–G728, 2005.[Abstract/Free Full Text]
  39. Matusiak D, Murillo G, Carroll RE, Mehta RG, Benya RV. Expression of vitamin D receptor and 25-hydroxyvitamin D3–1{alpha}-hydroxylase in normal and malignant human colon. Cancer Epidemiol Biomarkers Prev 14: 2370–2376, 2005.[Abstract/Free Full Text]
  40. McCoy JG, Avery DD. Bombesin: potential integrative peptide for feeding and satiety. Peptides 11: 595–607, 1990.[CrossRef][ISI][Medline]
  41. Mizejewski GJ. Role of integrins in cancer: survey of expression patterns. Proc Soc Exp Biol Med 222: 124–138, 1999.[Abstract/Free Full Text]
  42. Nakayamada S, Okada Y, Saito K, Tamura M, Tanaka Y. beta1 Integrin/focal adhesion kinase-mediated signaling induces intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation. J Biol Chem 278: 45368–45374, 2003.[Abstract/Free Full Text]
  43. Nakayamada S, Saito K, Fujii K, Yasuda M, Tamura M, Tanaka Y. beta1 Integrin-mediated signaling induces intercellular adhesion molecule 1 and Fas on rheumatoid synovial cells and Fas-mediated apoptosis. Arthritis Rheum 48: 1239–1248, 2003.[CrossRef][ISI][Medline]
  44. Narayan S, Guo JS, Townsend CM, Singh P. Specific binding and growth effects of bombesin-related peptides on mouse colon cancer cells in vitro. Cancer Res 50: 6772–6778, 1990.[Abstract/Free Full Text]
  45. Ng IO. Prognostic significance of pathological and biological factors in hepatocellular carcinoma. J Gastroenterol Hepatol 13: 666–670, 1998.[ISI][Medline]
  46. Nicolson GL, Moustafa AS. Metastasis-associated genes and metastatic tumor progression. In Vivo 12: 579–588, 1998.[ISI][Medline]
  47. Park YJ, Kim WH, Paeng SS, Park JG. Histoclinical analysis of early colorectal cancer. World J Surg 24: 1029–1035, 2000.[CrossRef][ISI][Medline]
  48. Patel O, Dumesny C, Giraud AS, Baldwin GS, Shulkes A. Stimulation of proliferation and migration of a colorectal cancer cell line by amidated and glycine-extended gastrin-releasing peptide via the same receptor. Biochem Pharmacol 68: 2129–2142, 2004.[CrossRef][ISI][Medline]
  49. Qin Y, Halmos G, Cai RZ, Szoke B, Ertl T, Schally AV. Bombesin antagonists inhibit in vitro and in vivo growth of human gastric cancer and binding of bombesin to its receptor. J Cancer Res Clin Oncol 120: 519–528, 1994.[CrossRef][ISI][Medline]
  50. Radulovic S, Miller G, Schally AV. Inhibition of growth of HT-29 human colon cancer xenografts in nude mice by treatment with bombesin/gastrin releasing peptide antagonist (RC-3095). Cancer Res 51: 6006–6009, 1991.[Abstract/Free Full Text]
  51. Rampaul RS, Pinder SE, Elston CW, Ellis IO. Prognostic and predictive factors in primary breast cancer and their role in patient management: The Nottingham Breast Team. Eur J Surg Oncol 27: 229–238, 2001.[CrossRef][ISI][Medline]
  52. Riecken EO, Rosewicz S. Retinoids in pancreatic cancer. Ann Oncol 10, Suppl 4: 197–200, 1999.[Abstract/Free Full Text]
  53. Spindel ER, Giladi E, Brehm P, Goodman RH, Segerson TP. Cloning and functional characterization of a complimentary DNA encoding the murine fibroblast bombesin/gastrin-releasing peptide receptor. Mol Endocrinol 4: 1956–1963, 1990.[Abstract]
  54. Sunday ME, Hua J, Reyes B, Masui H, Torday JS. Anti-bombesin monoclonal antibodies modulate fetal mouse lung growth and maturation in utero and in organ culture. Anat Rec 236: 25–32, 1993.[CrossRef][Medline]
  55. Tachimori A, Yamada N, Sakate Y, Yashiro M, Maeda K, Ohira M, Nishino H, Hirakawa K. Up regulation of ICAM-1 gene expression inhibits tumour growth and liver metastasis in colorectal carcinoma. Eur J Cancer 41: 1802–1810, 2005.[CrossRef][ISI][Medline]
  56. Uzdensky A, Kolpakova E, Juzeniene A, Juzenas P, Moan J. The effect of sub-lethal ALA-PDT on the cytoskeleton and adhesion of cultured human cancer cells. Biochim Biophys Acta 1722: 43–50, 2005.[Medline]
  57. Vainer B, Sorensen S, Seidelin J, Nielsen OH, Horn T. Expression of ICAM-1 in colon epithelial cells: an ultrastructural study performed on in vivo and in vitro models. Virchows Arch 443: 774–781, 2003.[CrossRef][ISI][Medline]
  58. Vanky F, Wang P, Patarroyo M, Klein E. Expression of the adhesion molecule ICAM-1 and major histocompatibility complex class I antigens on human tumor cells is required for their interaction with autologous lymphocytes in vitro. Cancer Immunol Immunother 31: 19–27, 1990.[CrossRef][ISI][Medline]
  59. Venaille TJ, Mendis AH, Warton A, Walker L, Papadimitriou JM, Robinson BW. Study of human epithelial cell detachment and damage: development of a model. Immunol Cell Biol 67: 359–369, 1989.
  60. Villavicencio H, Rubio-Briones J, Regalado R, Chechile G, Algaba F, Palou J. Grade, local stage and growth pattern as prognostic factors in carcinoma of the penis. Eur Urol 32: 442–447, 1997.[ISI][Medline]
  61. Wang LH, Coy DH, Taylor JE, Jiang NY, Moreau JP, Huang SC, Frucht H, Haffar BM, Jensen RT. des-Met carboxyl-terminally modified analogues of bombesin function as potent bombesin receptor antagonists, partial agonists, or agonists. J Biol Chem 265: 15695–15703, 1990.[Abstract/Free Full Text]
  62. Wimmenauer S, Keller H, Ruckauer KD, Rahner S, Wolff-Vorbeck G, Kirste G, von Kleist S, Farthman EH. Expression of CD44, ICAM-1 and N-CAM in colorectal cancer. Correlation with the tumor stage and the phenotypical characteristics of tumor-infiltrating lymphocytes. Anticancer Res 17: 2395–2400, 1997.[ISI][Medline]
  63. Wood CB, Gillis CR, Hole D, Malcom AJH, Blumgart LH. Local tumor invasion as a prognostic factor in colorectal cancer. Br J Surg 68: 326–328, 1981.[ISI][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/1/G182    most recent
00201.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Taglia, L.
Right arrow Articles by Benya, R. V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Taglia, L.
Right arrow Articles by Benya, R. V.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.