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Cell Growth & Differentiation Vol. 11, 385-393, July 2000
© 2000 American Association for Cancer Research


Articles

Gastrin-releasing Peptide Is a Mitogen and a Morphogen in Murine Colon Cancer1

Robert E. Carroll, Kristina A. Matkowskyj, Maria S. Tretiakova, James F. Battey and Richard V. Benya2

Departments of Medicine [R. E. C., K. A. M., M. S. T., R. V. B.], Pathology [K. A. M.], and Pharmacology [R. V. B.] University of Illinois at Chicago, and Chicago Veterans Administration Medical Center (West Side Division) [R. E. C., K. A. M., M. S. T., R. V. B.], Chicago, Illinois 60612, and National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, Maryland 20892 [J. F. B.]

Abstract

Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.

Introduction

The progression of normal colonic epithelium to adenocarcinoma is increasingly well established at the genetic and molecular level. Colon cancer is now appreciated to be attributable to both the mutational inactivation of a series of tumor suppressor genes (1, 2) as well as the activation of specific oncogenes (3) . However, the molecular derangements responsible for the pattern of cellular organization in colon cancer, or tumor grade, are not well understood.

Well-differentiated cancers phenotypically resemble the tissue type from which they originate, whereas poorly differentiated cancers display a chaotic and disorganized histology with few features of their tissue of origin. Most solid tumors display a near-uniform degree of differentiation, with increasing dedifferentiation inversely correlated with patient survival (4) . In contrast, adenocarcinomas of the colon are unusual insofar as multiple regions of varying differentiation are simultaneously present in larger tumors (5–7) . Yet the factor(s) regulating the differentiation of individual tumor cells in colon cancer, along with their contribution to cancer progression, have not been identified.

Neuropeptides have been widely studied as growth factors in many different cancer types, including those arising from the colon. Recent work, however, suggests that some of these peptide hormones may also play a role in the differentiation of normal and malignant tissues. GRP3 is such a hormone that has been shown previously to be important in the normal development of selected tissues such as the lung (8, 9) , as well as plays a role as a growth factor when aberrantly secreted by SCCL cells (10) . Within the gastrointestinal tract, GRP is normally found only in enteric nerve terminals (11–13) , where it acts to regulate intestinal motility by binding to a specific seven-transmembrane-spanning, G protein-coupled receptor (14) . In contrast, GRP and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed by many human colon cancers and cancer cell lines (reviewed in Ref. 15 ). All colon cancer cell lines expressing GRP-R proliferate when this receptor is activated (16–20) . Thus, GRP is commonly held to act as an autocrine growth factor when it and its receptor are aberrantly expressed. Yet our recent study of human colon cancers suggested that although GRP has mitogenic properties, this peptide hormone might have a more important role acting in an autocrine fashion as a morphogen (7) . Specifically, we found for the first time that all well-differentiated histological regions within colon cancer, irrespective of tumor stage, coexpressed both GRP and GRP-R. In contrast, GRP/GRP-R coexpression was never detected in any colon cancer region that was poorly differentiated or in any metastatic lesion. Thus, this study suggested that aberrant GRP/GRP-R expression might be important in maintaining colon cancers in a well-differentiated state.

To evaluate this hypothesis directly, we studied azoxymethane-induced colon cancers in wild-type GRP-R+/+ C57BL/6J mice and their recently created nullizygous GRP-R-/- counterparts (21) . Similar to what we have described in humans (7) , we show that although GRP and GRP-R are not normally expressed by epithelial cells lining the colon, both are aberrantly expressed by all better differentiated cells within cancers in wild-type mice. Significantly, colon cancers in wild-type mice became better differentiated as they increased in size, whereas tumors in GRP-R-/- mice underwent progressive dedifferentiation. Our findings therefore provide direct evidence that GRP is a morphogen, the first such agent identified to regulate colon cancer differentiation.

Results

Noncolonic Effects of AOM.
Although AOM has been well studied in rats (reviewed in Ref. 22 ), no published data exist reporting the effects of this carcinogen in this strain of mouse. In preliminary experiments, we attempted using higher concentrations of AOM (15 µg/mg) given weekly for only 2 weeks. Unfortunately, this dose did not induce the formation of colon cancers (n = 30; data not shown) but often resulted in the development of fulminant hepatic failure, as we have reported previously (23) . We therefore treated mice with the alternative protocol of lower dose but weekly s.c. injections of AOM (7.5 µg/mg). Similar to what has been reported previously for its chemical precursor dimethylhydrazine (24, 25) , even this lower dosing regimen resulted in significant side effects including intestinal ischemia and peritoneal hemangioendothelioma formation. The former occurred in ~25% of animals and could be recognized by the absence of expected weight gain, whereas the latter occurred in ~30%. The large cystic peritoneal hemangioendotheliomas could be identified by abdominal palpation or, because they often bled internally, by the presence of white paws reflective of anemia. Both complications occurred equally in wild-type and GRP-R-/- mice and with an incidence similar to that which has been recorded previously for dimethylhydrazine (24, 25) . Both conditions contributed to the increasing difficulty in obtaining tumors in animals who received AOM for >31 weeks, whereas it was impossible to maintain animals for >35 weeks.

Effect of AOM on Colonic Epithelia.
AOM caused equivalent increases in colonic crypt cell proliferation rates in both wild-type and GRP-R-/- mice. Specifically, whereas basal crypt proliferation rates were ~5 mitoses/HPF/animal in both wild-type and GRP-R-/- mice, AOM administration approximately doubled the number of metaphases/HPF/animal observed for as long as mice received this drug (Table 1Citation ). There was no statistically significant difference in the proliferation rates of nonmalignant crypts between wild-type and GRP-R-/- mice for as long as they continued to receive this drug (Table 1)Citation .


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Table 1 Comparison of wild-type and GRP-R knockout mice as a function of time of AOM exposure

Data expressed as means ± SE. Totals are for all animals in this study that received AOM for any length of time and specifically does not include those listed as controls (i.e., 0 wk).

 
Polyps did not develop until mice had received AOM for at least 21 weeks, whereas significant numbers of tumors were not observed until mice had received this drug for >25 weeks. In all instances, polyps and tumors developed within 3 cm of the anal verge. Overall, no significant difference between wild-type and knockout mice could be detected in terms of polyp or tumor location, number, or size (Table 1)Citation . However, and consistent with prior studies demonstrating that GRP is a mitogen, tumors developing in wild-type mice had a significantly higher proliferation index (19.1 ± 1.9 metaphases/HPF/tumor) than those arising in GRP-R-/- mice (6.5 ± 2.0 metaphases/HPF/tumor; P < 0.05; Table 1Citation ).

Evaluation of Tumors in Wild-Type Mice.
Similar to what we have described previously in humans (7) , GRP/GRP-R are not expressed by normal-appearing epithelial cells lining the colons of wild-type mice (Fig. 1ACitation ). In contrast, both proteins are expressed by all better differentiated adenocarcinomas developing in wild-type mice (Fig. 1, B and C)Citation . Similar to what has been observed in human colon cancer (5–7) , murine colonic tumors were histologically nonhomogeneous (Fig. 1DCitation ). Additionally, the amount of GRP and GRP-R detected immunohistochemically in colon cancers developing in wild-type mice was directly related to the degree of tumor cell differentiation (Table 2Citation ). Specifically, GRP/GRP-R expression was essentially undetectable in the most poorly differentiated tumor regions and was ~6–7-fold higher in well-differentiated tumor cells as compared with moderately poorly differentiated tumor cells (Table 2)Citation . Thus, colon cancers in AOM-treated mice are similar to tumors arising in humans insofar as both are histologically heterogeneous, with GRP/GRP-R expression strongly associated with the degree of tumor cell differentiation.



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Fig. 1. A, GRP immunoreactivity is not normally detected in epithelial cells lining the murine colon. Similar results are obtained for GRP-R immunopositivity. x100. All better differentiated colon cancers developing in wild-type C57BL/6J mice exposed to AOM aberrantly expressed GRP (B) and GRP-R (C). Immunohistochemistry was performed as described previously (7) ; x100. Insets in A–C, the same tissues processed as controls, whereby they were treated identically but were not exposed to primary antibody. D, similar to human colon cancers, murine tumors contained multiple distinct histological regions. One can appreciate the presence of well-differentiated (white arrowhead), moderately differentiated (green arrowheads), and poorly differentiated (yellow arrowhead) cells in a single HPF. H&E, x400.

 

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Table 2 GRP and GRP-R expression in colon cancers developing in wild-type mice as a function of tumor cell differentiation

Overall, 194 separate regions of distinct differentiation were identified in 12 tumors that developed in GRP-R+/+ wild-type mice. Data expressed as means ± SE.

 
Consequence of GRP-R Gene Elimination on Tumor Histology.
To determine the relative percentage tumor cells of each differentiation type occupied, we first acquired images of the entire cancer cross-section at x100 using a 9-million pixel digital scanning camera. Using the software program Photoshop, we then determined the total number of pixels, a unit measure of area, occupied by cells of defined differentiation and expressed this number relative to the area occupied by the entire cancer (technique illustrated in Fig. 2Citation , top panel). In this manner, we could determine that despite the fact that GRP-R-/- mice were incapable of expressing this protein, no difference in histology could be detected between small and presumably younger tumors developing in mice of either genotype. For animals of either genotype, small tumors were primarily moderately differentiated, although some heterogeneity in overall histopathology was apparent (Fig. 2Citation , bottom left panel). In contrast, larger and presumably older cancers in wild-type mice became significantly better differentiated (Fig. 2Citation , bottom right panel; Fig. 3ACitation ), whereas tumors in GRP-R-/- mice progressively dedifferentiated (Fig. 2Citation , bottom right panel; Fig. 3BCitation ), than observed in the smaller/earlier lesion.



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Fig. 2. Relative amount of tumor of the indicated differentiation type in small and large murine adenocarcinomas. Top panel, individual tumor cell regions were identified as shown within the bold tracings, and the area occupied in pixels was determined using the Magic Wand function in Adobe Photoshop. The sum total area of each differentiation type was then expressed relative to the area occupied by the entire tumor. We compared all tumors <7.5 mg (n = 11; bottom left panel) or >15 mg (n = 8; bottom right panel). In all instances, images were acquired at x100 using a 9-million pixel Microlumina Ultra Resolution Scanning Digital Camera (Leaf Systems). The diamonds ({diamondsuit}) within the box straddling the lower panels identifies the mean differentiation, with closed symbols for wild-type tumors and open symbols for GRP-R knockout tumors. Data expressed are means; bars, SE.

 


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Fig. 3. Larger tumors in wild-type mice became significantly better differentiated (A) than those developing in GRP-R-/- mice (B). H&E, x400. Tumors from wild-type mice (C) expressed fewer mucincontaining glands as compared with those from GRP-R-/- mice (D). Mucin is identified by the carmine red color. Sections were counterstained with modified Mayer’s Mucicarmine as described previously (53) . x40.

 
Larger tumors in GRP-R-/- mice also developed prominent mucin-filled glands (Fig. 3DCitation ) that were not observed in tumors developing in wild-type mice (Fig. 3CCitation ). Although small tumors (i.e., <7.5 mg) from GRP-R-/- mice did not show evidence of mucinous glands, ~15% of intermediate-sized tumors (7.5–15.0 mg) and ~80% of larger tumors (>15.0 mg) developed these structures.

Tumor Proliferation Rates Vary with the Extent of GRP/GRP-R Expression.
We investigated whether the change in histomorphology between small and large cancers from wild-type and GRP-R-deficient mice could be attributable to alterations in tumor cell proliferation. Although better differentiated tumors had higher proliferation rates in both wild-type and GRP-R-/- mice, these rates were significantly higher in tumors expressing increased amounts of GRP and GRP-R (Fig. 4Citation ). Thus, GRP/GRP-R expressing cancers possess a growth advantage that might partially account for their histological improvement as they increased in size in wild-type mice (Fig. 2)Citation . The better differentiated tumor regions in GRP-R-/- mice also contained more proliferating cells than less well-differentiated tumors (Fig. 4)Citation , suggesting the presence of other differentiation-associated mitogens. These factor(s), however, were not sufficient to provide these histological regions with a significant growth advantage because tumors in GRP-R-deficient mice progressively dedifferentiated with increasing size (Fig. 2Citation , right panel). Intriguingly, and despite higher numbers of proliferating cells in tumors from wild-type mice as compared with tumors in GRP-R-/- mice, no significant difference in tumor size could be detected (Table 1)Citation .



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Fig. 4. Cellular proliferation as a function of differentiation status in tumors from wild-type () and GRP-R-/- ({square}) mice. Individual tumor cells were manually counted and graded in 15 separate tumors of varying size. Metaphase complexes were identified using the criteria of Baak (50) and expressed as the percentage of total cells counted per differentiation type. Overall, 1806 cells in wild-type tumors and 2248 cells in GRP-R-/- tumors were counted. Total GRP and GRP-R expression in tumors developing in wild type mice (line), as determined by quantitative immunohistochemistry, reflects the summed amounts from Table 2Citation . Bars, SE.

 
Evaluation of Tumor Apoptosis Rates.
Despite the greater number of proliferating cells in tumors from wild-type as compared with GRP-R-deficient mice, tumor size/weight was the same in animals of either genotype exposed to AOM for identical lengths of time. This suggested that tumors in wild-type mice might have higher rates of cell turnover. To determine whether this was the case, we systematically determined the number of apoptotic cells in tumors from wild-type and GRP-R-/- mice. By modified terminal deoxynucleotidyl transferase-mediated nick end labeling assay, there were 4.4 ± 1.0 apoptotic cells/HPF in tumors from wild-type mice, whereas there were but 1.6 ± 0.3 apoptotic cells/HPF in tumors from GRP-R-/- mice (P < 0.05). This ~3-fold increase in apoptosis was similar to the degree to which tumor cell proliferation was increased in cancers developing in wild-type as compared with GRP-R-/- mice (Table 1)Citation . Thus, the increase in overall cell proliferation observed in tumors from wild-type mice is balanced by a similarly increased rate of cell apoptosis, accounting for the similar size of cancers developing in mice of either genotype when exposed to AOM for identical lengths of time.

GRP Increases FAK Expression.
To examine a potential mechanism whereby GRP-R could influence tissue morphology, we investigated expression of FAK. To do this, we performed quantitative immunohistochemistry to determine the amount of FAK present in each region of distinct differentiation contained within each tumor. FAK expression was significantly lower in tumors from knockout mice as compared with those from wild-type mice (Fig. 5Citation , left panel), with higher FAK levels seen in better differentiated tumor regions. This difference was observed even when regions of similar histology were compared between wild-type and knockout mice (Fig. 5Citation , left panel).



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Fig. 5. Expression of FAK (left panel) and c-Src (right panel) in colon cancers developing in wild-type mice () or GRP-R-/- mice ({square}) as a function of differentiation. Quantitative immunohistochemistry was performed to determine the amount of FAK or c-Src chromogen, with values recorded as energy units per pixel (eu/pix). For each differentiation type, the amount of chromogen was determined in 10 separate regions for tumors developing in wild-type and GRP-R knockout mice, respectively. Data are expressed as means; bars, SE.

 
To evaluate whether this difference in FAK expression was specific, we also studied cellular expression of c-Src. Similar to GRP and its receptor, recent reports have indicated that c-Src expression varies as a function of tumor cell differentiation (26) . By quantitative immunohistochemistry, we found that c-Src was present in greater amounts in well-differentiated as compared with poorly differentiated tumor cells (Fig. 5Citation , right panel). Yet in contrast to what was observed for FAK, similar high levels of c-Src were detected in better differentiated cancer regions in tumors developing in mice of either genotype. Thus, these data suggest, but admittedly do not prove, that FAK expression may not simply be a function of tumor cell differentiation but rather that FAK expression might be regulated as a function of GRP receptor expression.

Discussion

GRP and its pharmacological homologue bombesin are well known to cause the proliferation of a wide range of cell types including those derived from colon cancers (16–20) , normal human bronchial epithelial cells (27) , pancreatic adenocarcinomas (28) , and a number of breast cancer cell lines (29–31) . Arguably the best studied are those derived from human SCCL, most of which secrete GRP, express GRP-R, and respond to exogenous agonist by proliferating in a concentration-dependent manner (10, 32, 33) . The action of GRP as an autocrine growth factor in these cells is specific as antibodies and antagonists block their growth both in vitro and in vivo (10) . Similarly, antagonists can attenuate the growth of gastrointestinal cancer cell lines expressing GRP-R (16–20) . Thus, these studies clearly demonstrate that GRP causes cell proliferation. Yet increasing evidence suggests that the mitogenic properties of GRP may be subordinate to it acting as a morphogen in the few cancers in which this peptide hormone has been studied carefully.

Most, but not all, SCCLs express GRP-R. Recent studies indicate that GRP-Rs are only present in well-differentiated (or typical), but not in poorly differentiated (or variant), small cell carcinomas (34–36) . Likewise, GRP-Rs are not normally found in prostatic tissues (37) but are up-regulated in prostate cancer, with highest receptor concentrations observed in well-differentiated as compared with poorly differentiated tumors (38) . Finally, we showed recently in human colon cancer that GRP/GRP-R coexpression occurs independently of tumor stage and is related to the degree of tumor differentiation. Although colon cancers differ from most solid tumors insofar as they are histologically heterogeneous, we observed that these two proteins were only coexpressed by well-differentiated tumor regions whereas poorly differentiated tissues never coexpressed GRP/GRP-R (7) . In aggregate, therefore, these observations suggested the possibility that GRP and its receptor might be important in regulating the differentiation of various cancers.

The role for GRP acting as a morphogen in colon cancer is conclusively demonstrated in the present study of wild-type C57BL/6J mice and GRP-R-/- mice. We show that whereas small, early tumors in mice of either genetic background were moderately differentiated, they became progressively better differentiated in wild-type mice but degenerated into poorly differentiated mucinous adenocarcinomas in GRP-R-/- mice. This is particularly interesting because mucinous adenocarcinomas are relatively rare in humans, where they are associated with a poor prognosis (39) and have a higher metastatic potential (40) , than nonmucinous tumors.

Yet our data also support GRP acting to cause cell proliferation, albeit weakly and without affecting overall tumor size. Tumors developing in wild-type mice contained ~3-fold more metaphase complexes than those in GRP-R-/- mice (Table 1)Citation , with the degree of cell proliferation related to the amount of GRP/GRP-R expressed (Fig. 4)Citation . Yet this increase in proliferation was almost precisely balanced by an increase in cellular apoptosis rates in tumors developing in wild-type as compared with GRP-R-/- mice. As a consequence, there was no difference in the size of tumors developing in mice of either genotype (Table 1)Citation . Indeed, if anything, mean tumor weight was greater in GRP-R-/- mice than in wild-type mice, although this difference was not statistically significant.

To identify a potential mechanism whereby GRP/GRP-R could act to regulate cancer appearance, we studied tumor cell FAK expression because recent studies have suggested that this enzyme is important in regulating the differentiation of cancers arising in the human gastrointestinal tract (41–43) . Specifically, whereas epithelial cells lining the human gastrointestinal tract do not normally express FAK, FAK expression is observed in cancers arising from the stomach and colon (43) . Although not directly commented upon in that particular study, FAK was only present in well-differentiated regions within any individual colon cancer. This observation is supported by recent in vitro investigations, where FAK expression in human colon cancer cell lines increased as they became better differentiated (41, 42) . This is particularly important because GRP has been shown to increase FAK expression in a number of cell lines (44–46) . In the current study, we showed that FAK expression was significantly decreased in tumors from GRP-R-/- mice, whereas such expression varied as a function of differentiation and GRP/GRP-R expression in tumors from wild-type mice (Fig. 5)Citation . This finding raises the possibility that the morphogenic capabilities of GRP may be mediated via GRP-R activation of FAK.

Two limitations of this study are that we only assessed the contribution of the GRP-R to tumor cell differentiation, and that this study did not determine the contribution(s) GRP/GRP-R make to other aspects of tumor behavior, including metastasis and animal survival/outcome. With respect to the first criticism, there are at least three different receptors in the bombesin family, and there is some evidence that subtypes other than the GRP-R can be up-regulated in selected cancers, such as those developing in the lung (47) . However, we are unaware of any prior study systematically investigating the expression of non-GRP bombesin receptor subtypes in colon cancer. Although we do not exclude the possibility that other bombesin receptors may also be important in regulating colon cancer differentiation, the present data do show that expression of at least the GRP-R subtype regulates tumor grade.

With respect to the second criticism, it should be observed that this study was not designed to study the ability of GRP/GRP-R to alter tumor behavior, such as metastasis or alter animal outcome, when aberrantly expressed in colon cancer. This is attributable to the fact that AOM, similar to its chemical precursor dimethylhydrazine, is associated with significant side effects including intestinal ischemia and peritoneal hemangioendotheliomas. These two complications occurred in ~50% of mice of either genotype treated with AOM and developed with increasing time of exposure to this carcinogen. Because tumors did not reliably develop until mice had been exposed to AOM for >25 weeks, it simply was not possible to determine whether the ability to express GRP-R altered important clinical end points, such as tumor metastases and/or animal survival. Because tumor differentiation inversely correlates with clinical outcome in cancers that are histologically homogeneous (4) , our findings suggest the possibility that GRP/GRP-R expression may be beneficial because these proteins promote the assumption of a well-differentiated phenotype. As such, aberrant GRP/GRP-R expression may prove to be of favorable prognostic significance.

Materials and Methods

Materials.
Wild-type C57BL/6J and GRP-R-/- mice were obtained by establishing breeding pairs as described previously (21) . To minimize potential hormonal influences, only male mice were studied. Because the murine GRP-R gene is located on the X chromosome (14) , male heterozygous (+/-) animals could not be evaluated. The University of Illinois at Chicago Animal Care Committee approved this study, and all care was provided in accordance with institutional guidelines. The source and use of antibodies for GRP and GRP-R were as described previously (7) ; antibody for FAK was from Upstate Biotechnology (Lake Placid, NY); antibody for c-Src was from Santa Cruz Biotechnology (Carpinteria, CA), whereas all other immunohistochemical materials were from Dako (Santa Cruz, CA). All other reagents including AOM and vincristine were from Sigma Chemical Co. (St. Louis, MO).

Colon Cancer Induction and Determination of Cell Proliferation.
Wild-type and GRP-R-/- mice received weekly s.c. injections of AOM at a dose of 7.5 µg/mg in normal saline as described previously (22) . Crypt and tumor proliferation rates were determined by administering 1 µg/mg vincristine 2 h prior to sacrifice. The stathmokinetic effects of vincristine are attributable to its arresting dividing cells in metaphase so that they can be readily identified and counted (48) . Crypt proliferation rates were determined by staining a segment of colon in Carnoy’s fixative for 4 h, dissecting out 20 separate colonic crypts, and counting the total metaphases as described previously (49) . The tumor proliferation index was determined by averaging the number of metaphases for 10 separate HPFs (x400), scoring only tumor sections meeting the criteria of Baak (50) .

Digital Image Capture and Assessment of Tumor Differentiation Status.
Digital images were acquired using a Microlumina Ultra Resolution Scanning Digital Camera [3380 x 2700 pixels (Leaf Systems, Fort Washington, PA)] attached to a Nikon E600 microscope system. Assessment of tumor differentiation was performed using a five-grade classification system based on what has been described previously (51) . 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. The intermediate grades of moderately well and moderately poor shared histological features of both well and moderate differentiation, or of moderate and poor differentiation, respectively.

Three representative 5-µm-thick sections were taken from the part of each tumor containing the largest cross-sectional area. After staining with H&E according to standard techniques, the entire tumor section was photographed at x100 using a 3380 x 2700 pixel Microlumina Ultra Resolution digital scanning camera (Leaf Systems). The number of pixels in each region of indicated histology in the entire tumor was determined using the Magic Wand function in Photoshop (Adobe, San Jose, CA) and then expressed relative to the area (i.e., in pixels) occupied by the entire tumor section. Each section was evaluated separately by two individuals (R. E. C., K. A. M.) at different times, and their values were averaged.

Quantitative Immunohistochemistry.
All immunohistochemistry was performed using a standard three-stage indirect immunoperoxidase technique on 5-µm-thick tissue sections. In all instances, sections were stained with the indicated antibody, incubated with biotinylated anti-rabbit IgG, followed by Streptavidin conjugated to horseradish peroxidase. Incubating slides with Liquid DAB Substrate-Chromogen System for 2 min identified bound antibody. All sections were counterstained using a 50% dilution of Gill’s hematoxylin.

Chromogen quantification was performed as described previously (52) . In brief, chromogen abundance was determined by calculating the cumulative signal strength within three regions for each separate histological region/tumor. For any image file of dimensions N1 by N2 pixels, and where n1 and n2 identify the specific position of the data within the digital image file, the cumulative signal strength (or mathematical energy, E) is defined as:

Thus, chromogen/pixel can be quantified by subtracting the energy of the control slide (i.e., not exposed to primary antibody) from that in the homologous regions of the experimental slide (i.e., exposed to primary antibody).

Tumor Apoptosis Rates.
Cellular apoptosis rates were determined using the Dead End Colorimetric Apoptosis Detection System (Promega Corp., Madison, WI), a modified terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Briefly, tumor sections were permeabilized using proteinase K, followed by labeling of DNA strand breaks with biotinylated nucleotide mix. After quenching background with 0.3% H2O2, tissues were treated with Streptavidin HRP and developed by exposing to 3,3'-diaminobenzidine for 10 min. Apoptotic cells/HPF (x400) were counted and averaged for a minimum of 10 HPFs/tumor.

Footnotes

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.

1 This work was supported by NIH Grant CA-80360 (to R. E. C.) and NIH Grant DK-51168 and a Veterans Affairs Merit Review Award (to R. V. B.). Back

2 To whom requests for reprints should be addressed, at Department of Medicine, University of Illinois at Chicago, 840 South Wood Street (M/C 787), Chicago, IL 60612. Phone: (312) 666-6500, extension 3439; Fax: (312) 455-5877; E-mail: rvbenya{at}uic.edu Back

3 The abbreviations used are: GRP, gastrin-releasing peptide; GRP-R, GRP receptor; AOM, azoxymethane; HPF, high-powered field; FAK, focal adhesion kinase; SCCL, small cell cancer of the lung. Back

Received for publication 3/16/00. Revision received 5/12/00. Accepted for publication 5/15/00.

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