Abnormalities in the function of receptor tyrosine kinases (RTKs) have been demonstrated to be important in the pathogenesis of cancer. H-Ryk, a new member of the RTK family, is an unusual RTK in that it is catalytically inactive because of amino acid substitutions of conserved residues in the catalytic domain. We show by immunohistochemistry that it is expressed in the epithelium, stroma, and blood vessels of normal tissues. Evaluation of a panel of 33 primary ovarian tumors (2 benign, 8 borderline, and 23 malignant) was performed. H-Ryk was overexpressed in borderline and malignant ovarian tumors. In serous and clear cell subtypes, there was increased expression in the epithelium, stroma, and blood vessels. Consistent with this observation, overexpression of H-Ryk in the mouse fibroblast cell line NIH3T3 induces anchorage-independent growth and tumorigenicity in nude mice. This implies that overexpression of the receptor can be transforming and may therefore be significant in the pathogenesis of ovarian cancer.

Aberrant signaling can arise as a consequence of overexpression (1), mutation (2), or translocation (3) of RTKs,3 which mediate the transmission of biological signals from the extracellular environment to the inside of the cell. However, the predominant RTK-related genetic alteration in cancer is amplification or overexpression. Diverse tumorigenesis associated with overexpression of the hepatocyte growth factor and its receptor, c-met, has been demonstrated in transgenic mice (4). Consistent with the amplification and/or overexpression of the c-erbB2 gene in a number of human tumors (1), it has been demonstrated that c-erbB 2 is a potent oncogene when overexpressed in NIH3T3 cells (5). The evidence derived from studies of animal models, tumor cell lines, and actual human tumors linking RTKs to the pathogenesis of ovarian cancer has become increasingly compelling (1, 6, 7). Ovarian carcinomas express many different growth factor receptors that mediate responses to a wide variety of growth factors expressed by the tumor cells themselves or host stromal tissue (8). To investigate a similarly critical role for as yet unidentified receptors in ovarian cancer, we isolated the H-Ryk receptor from an ovarian cancer cDNA library (9) in addition to others from different sources (10, 11, 12, 13). The most notable feature of the receptor are alterations to some of the most highly conserved residues in the catalytic domain, which result in impaired tyrosine kinase activity (10, 12). We report, for the first time, analysis of H-Ryk expression in normal tissues and ovarian tumors. In addition, we demonstrate that overexpression of H-Ryk in NIH3T3 confers transforming ability in vitro and in vivo. This observation is interesting because H-Ryk is catalytically inactive and belongs to a group of distinct receptors (c-erbB 3, CCK-4, and Mep) with impaired tyrosine kinase activity (14).

Cell Culture and Transfection.

NIH3T3 mouse fibroblast cells were cultured in DMEM supplemented with 10% FCS. The PEO4 epithelial ovarian cancer cells were maintained in DMEM supplemented with 15% FCS, 1% nonessential amino acids, and 1% insulin. Both cell lines were cultured at 37°C in a humidified environment containing 5% CO2. The full-length H-Ryk cDNA (9) was cloned in the EcoRI site downstream of the cytomegalovirus promoter in the vector pcDNA3. The high efficiency calcium phosphate transfection method was used to transfect the NIH3T3 cell line, as described previously (15).

Immunoprecipitation and Immunoblotting.

For immunoprecipitation, cells were grown to 90% confluence and lysed on ice for 30 min in 1.00 ml of lysis buffer [1% Triton X-100, 0.5% NP40, 150 mm NaCl, 50 mm Tris-HCl (pH 7.4), 1 mm EDTA, and 0.5 mm EGTA] containing freshly added protease inhibitors (10 μg/ml aprotinin, leupeptin, pepstatin A, trypsin, and 100 μg/ml phenylmethylsulfonyl fluoride). Insoluble material was removed by centrifugation (30 min, 15 000 rpm, 4°C). Immunoprecipitation and immunoblotting was carried out with an antibody raised against a COOH-terminal peptide of H-Ryk, as described previously (9).

Anchorage Independence Assay.

A viable count of the cells was carried out by mixing 1 ml of the cell suspension with 1 ml of Nigrosine. Viable cells were identified by their ability to exclude the Nigrosine dye. The soft agar assay was done in 0.2% agar, DMEM, and 10% FCS and plated on a base of 1% agar, DMEM, and 10% FCS. Cells were seeded at a density of 1 × 104 per 35-mm plate in soft agar containing 10% FCS. Colonies with >50 cells, 100 μm in diameter, were scored after 3 weeks. Each assay was performed in triplicate on two independent occasions.

In Vivo Studies.

Female nude (strain, Nu/nu) mice were bred at Imperial Cancer Research Fund Clare Hall Laboratories (London, United Kingdom) and received injections at 8 weeks. NIH3T3 H-Ryk transfected cells were harvested by adding trypsin/versene and then washing them in PBS. The cells were centrifuged and counted, and 1 × 107 cells were resuspended in 0.2 ml of PBS prior to being injected s.c. into the flank using a 1-ml syringe and 21-gauge needle. For inoculations in which Matrigel was to be added, 1 × 107 cells were resuspended in 0.2 ml of PBS chilled on ice and then made up to 0.4 ml with Matrigel before being similarly injected. After inoculation, mice were maintained under observation. The mice were monitored for growth of tumors and measured twice a week from the time of appearance. When the tumors reached 12 mm, mice were killed, and their tumors were removed, cut into 2–3 mm pieces, and stored in liquid nitrogen for subsequent studies.

Immunohistochemistry.

Tissue sections were cut at 4 μm onto Vectabond (Vector Laboratory)-coated slides and incubated at 37°C overnight. They were dewaxed and treated with 3% hydrogen peroxide in distilled water for 20 min to block endogenous peroxidase. For antigen retrieval, the sections were placed for 90 s in a domestic pressure cooker containing a boiling solution of 0.01 m sodium citrate. The polyclonal rabbit antibody, H-Ryk 15.2, was applied to the sections at a concentration of 5 μg/ml in Tris-buffered saline (pH 7.6). They were incubated for 1 h at room temperature. After three washes in TBS over 10 min, goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase (DAKO) at a dilution of 1:100 in TBS was applied to the sections for 30 min at room temperature. The peroxidase was visualized using hydrogen peroxide as the substrate and diaminobenzidine tetrahydrochloride as the chromogen. The sections were counterstained with hematoxylin and mounted in a xylene-based solution. For the negative control, a 10-fold excess of competing 15.2 cognate peptide was used.

Immunostaining of Normal Tissues.

H-Ryk immunoreactivity was detected in all normal tissues analyzed with the exception of the prostate, where receptor expression was not detected (Table 1). Surface epithelium, particularly of the small bowel, breast, tonsil, thyroid, and fallopian tube showed moderate to high expression of the H-Ryk RTK (Fig. 1). In the small bowel, the expression was most marked at the villous tips. In the uterus, immunoreactivity was absent in proliferative endometrium, but mild reactivity was present in the secretory phase. In the breast, immunoreactivity was mostly noted in lobular acini and ductal epithelium (Fig. 1 C). In addition to the surface epithelium, expression of H-Ryk was also pronounced in smooth muscle cells. This was most prominent in the myometrium and vas deferens. The smooth muscle cells of blood vessels, notably of the brain and heart, had moderate to high levels of H-Ryk expression. Overall, expression of H-Ryk was prominent in the epithelial and stromal compartments in a pattern distinctive for each tissue.

Immunostaining of Normal Ovary and Ovarian Tumors.

In the normal ovary, low expression of the H-Ryk receptor was detected in the epithelium with moderate expression of the receptor being detected in the blood vessels (Fig. 2, A and B). Expression of H-Ryk was increased (+++) in serous borderline tumors in epithelium, stroma, and blood vessels (Table 2). Malignant serous and clear cell tumors had a higher frequency of overexpression of H-Ryk compared with mucinous and endometrioid tumors. However, there was no obvious correlation of H-Ryk expression with the grade of the tumor. Immunoreactivity was also present in the stromal tissue of most tumors, predominantly in the desmoplastic tissue of carcinomas and in benign neoplastic stroma of borderline and benign serous tumors. Marked stromal expression of the receptor was observed in fibroblasts and inflammatory cells which were predominantly lymphocytic in type (Fig. 2, C and D). There was a slight tendency toward increased expression in the stroma of serous tumors compared with endometrioid, mucinous, and clear cell tumors. Endothelial and vascular smooth muscle immunoreactivity was seen in most tumors with moderate to high levels of expression in the serous neoplasms (Fig. 3).

Stable Transfection of H-Ryk in PEO4 and NIH3T3 Cells.

To investigate whether overexpression of H-Ryk results in cell transformation, we stably transfected the epithelial ovarian cancer cell line, PEO4, and the mouse fibroblast cell line, NIH3T3, with the H-Ryk cDNA. Ten PEO4 and 11 NIH3T3 stable clones were screened for expression of H-Ryk using a COOH-terminal H-Ryk antibody (9). A specific H-Ryk protein of Mr 110,000 was observed in the transfectants (Fig. 4). This recognition was specifically blocked by preabsorption of the antibody with the cognate 15.2 peptide. Densitometric measurements of the three PEO4 stable cell lines selected, PEO4:H-Ryk 20, 21, and 41, demonstrated approximately identical levels of receptor expression (ratio in arbitrary units; 1.40, 1.25, and 1.00 for transfectants 20, 21, and 41, respectively). Densitometric determination of the autoradiogram revealed that the levels of the NIH3T3:H-Ryk transfectants were ≈9-fold higher than those detected in the parental cell line (ratio in arbitrary units of 1.00, 1.10, 6.70, and 9.00 for the parental NIH3T3, NIH3T3:pcDNA3, NIH3T3:H-Ryk 13, and NIH3T3:H-Ryk 4, respectively).

Overexpression of H-Ryk Is Transforming in Vitro and in Vivo.

The H-Ryk transfectants showed no gross morphological alterations but proliferated at a much faster rate compared with the vector-transfected PEO4 or NIH3T3 cell lines in medium supplemented with either 10 or 0.1% FCS (data not shown). The H-Ryk transfectants were able to form colonies in soft agar at high efficiency in contrast to the parental cell lines transfected with the vector. In comparison with the vector-transfected PEO4 cell line, the PEO4:H-Ryk stable cell lines displayed an increased ability to proliferate indefinitely on soft agar (Fig. 4,C). The NIH3T3:H-Ryk 4 transfectant showed higher clonogenic affinity in the soft agar assay than the NIH3T3:H-Ryk 13 transfectant (Fig. 4 D). This is probably due to the fact that NIH3T3:H-Ryk 4 had higher levels of the receptor compared with NIH3T3:H-Ryk 13, consistent with the higher levels of H-Ryk expression in NIH3T3:H-Ryk 4, as determined by densitometric analysis of both transfectants.

We next tested the ability of the H-Ryk transfectants to induce tumorigenicity in vivo. In the group of mice that underwent s.c. xenografting without Matrigel, the nodules of s.c. tumor were first palpable at days 9 and 13 after initial inoculation of the NIH3T3:H-Ryk transfectants 4 and 13, respectively. s.c. injections of the NIH3T3 transfectants consistently resulted in the development of tumors at the site of injection with a latency of 4–5 weeks (Fig. 4, E and F). However, there was no evidence of metastasis in the majority of the mice analyzed with the exception of one mouse, which had an i.p. tumor and some i.p. fluid. In the group of mice that underwent s.c. injection of the transfectants with Matrigel, tumor growth was accelerated with a significant reduction in latency from 4 to 2 weeks. For both transfectants, the s.c. xenografts consisted of a multiple nodular mass with a latency period of 2 weeks (10 of 10 mice). Although the latency period was significantly reduced, there was still no evidence of metastasis at autopsy. Mice injected with vector-only control NIH3T3 transfected cells did not develop any tumors, even after prolonged observation (6 months). In contrast to the in vitro transforming ability of the PEO4:H-Ryk transfectants, no tumor formation was observed after s.c. transplantation of the PEO4:H-Ryk stable cell lines in the presence or absence of Matrigel (6 months).

Medium- to high-grade tumors were observed on histological examination of the NIH3T3:H-Ryk nude mice tumors. The morphology of the tumors was that of a fibrosarcoma with interlacing irregular bundles of malignant fibroblastic cells. Areas of necrosis, possibly due to ischemia (Fig. 4,G), were seen at the edges and in the center of some tumors. Tumor infiltration into skeletal muscles was also apparent (Fig. 4,H). Most cells were spindle shaped and contained hyperchromatic, pleomorphic nuclei (Fig. 4,I). Analysis of the proportion of cells undergoing mitosis (mitotic index) indicated that the tumors were variably mitotically active. Marked collagen formation was also observed in some of the tumors analyzed (Fig. 4 J). There were no appreciable differences between mice, although some variation in mitotic activity and pleomorphism between and within the tumors was apparent.

The H-Ryk RTK was expressed in all of the normal tissues analyzed with the exception of the prostate. Most of the H-Ryk protein was expressed in the epithelium or the stroma in a pattern distinctive for each tissue. In the stroma, expression was prominent in the smooth muscle cells, particularly of the blood vessels and vas deferens. The expression of H-Ryk in the stromal compartment of various tissue types suggests a role for the receptor in cell-cell interactions. The presence of putative leucine-rich motifs in the extracellular domain of H-Ryk supports this hypothesis. The ubiquitous expression of H-Ryk correlates with the mRNA data (9, 10, 11, 12, 13). The moderate to high expression of H-Ryk in the smooth muscle cells is consistent with the abundance of H-Ryk message in the muscle (9) and the role of the Drosophila Ryk homologue, derailed (drl), in muscle attachment (16). derailed has also been implicated in neuronal pathway selection in Drosophila (17). Whether H-Ryk plays a similar role in mammals remains to be determined. However, low expression of H-Ryk in the brain suggests that the receptor may not have an analogous function in humans, although temporal expression of the receptor during brain development cannot be discounted.

Defining the molecular mechanisms of oncogene-dependent cellular transformation is critical for the understanding and treatment of human cancer. A number of in vitro experiments have demonstrated that increased expression of some proto-oncogenes can lead to neoplastic transformation (4, 5). In naturally occurring tumors, increases in gene expression have been postulated to occur through a number of mechanisms, which include DNA amplification or rearrangement. In several human tumors, increased expression of proto-oncogenes apparently takes place in the absence of genetic changes at the proto-oncogene locus, and these changes have been suggested to play an active role in tumor formation (18). However, in the absence of such structural alterations, it is difficult to know whether increased expression of a proto-oncogene is related to the tumorigenic process or reflects the abnormal growth status present in the tumor. H-Ryk maps to chromosome 3q22–24, and no genetic alterations at this locus have been reported in ovarian cancer.4 H-Ryk epithelial and stromal expression in normal ovary was negligible, indicating that the basal expression of the receptor in normal ovary is low. In the ovarian adenocarcinomas, overexpression of H-Ryk was observed in 50% of cases analyzed. The high levels of expression of H-Ryk receptor in clear cell and serous neoplasms suggest that the receptor may play an important role in the pathogenesis of these subtypes of ovarian neoplasm. In addition, the moderate to high levels of H-Ryk expression in the benign and borderline serous neoplasms imply that H-Ryk overexpression may be an early event in the development of serous adenocarcinomas. Although the number of tumors analyzed is small, these findings provide the first evidence that H-Ryk overexpression may play a role in ovarian tumorigenesis. Future evaluation of a larger panel of tumors will determine whether up-regulation of H-Ryk is an independent prognostic factor or if the receptor acts cooperatively with other cellular oncogenes in the transformation of the ovary.

Anchorage-independent growth is the in vitro characteristic that correlates best with tumorigenicity in vivo(19). Normal cell growth requires cell adhesion to extracellular matrix proteins as well as stimulation by growth factors. Conversely, cellular transformation leads not only to serum-independent growth but also to anchorage-independent growth. This study demonstrates that H-Ryk-transfected cells have the ability to display serum-independent growth and proliferate indefinitely in semisolid agar. Consistent with this finding was the ability of the NIH3T3:H-Ryk transfectants to induce tumor formation in nude mice. The tumors appeared with a short latent period and grew rapidly, thereby excluding the possibility that secondary alterations selected in vivo were responsible for the tumorigenic phenotype of these cells. This is supported by the inability of the control vector transfectant to form any tumors. There were differences observed between the NIH3T3:H-Ryk transfectants transplanted in the presence or absence of Matrigel, as demonstrated by a significantly shorter latency period for the Matrigel transplantation. This is because Matrigel is rich in extracellular matrix proteins and growth factors, which confer an advantage in the establishment of xenografts from a variety of cell lines (20). In contrast, no tumor formation was observed in the nude mice after s.c. transplantation of the PEO4:H-Ryk transfectants. To date, a number of human ovarian cancer cell lines have been established. However, a limited number have the capacity to be tumorigenic in nude mice. This has been attributed to the fact that s.c. implantation of ovarian tumor cell lines results in low uptake rates (21). Recent studies have also indicated that cell suspensions may not express the full metastatic potential of the original tumor (22, 23). It is possible that any of the these factors might have contributed to the inability of the PEO4:H-Ryk transfectants to induce tumor formation. The development of more reproducible models of human ovarian cancer that involve either i.p. (24) or orthotopic (25) transplantation of cell suspensions into nude mice provides an alternative means of validating whether overexpression of H-Ryk in ovarian epithelial cells is sufficient to induce ovarian tumorigenesis.

There are two possible hypotheses to explain how mere overexpression results in transformation: (a) because the ligand for H-Ryk has not yet been identified, it is possible that H-Ryk-transformed cells themselves might produce the ligand, thus leading to chronic stimulation of the receptor; and (b) at elevated receptor levels, an increased formation of receptor dimers may occur, even in the absence of ligand, resulting in constitutive stimulation of the receptor and aberrant signaling. The fact that the H-Ryk receptor contains alterations to some of the highly conserved amino acid residues in the catalytic domain that account for the impaired tyrosine kinase activity makes this transforming ability all the more interesting. This is by no means unique because it has been reported recently that the catalytically impaired receptor tyrosine kinase, Cck-4, may be involved in the pathogenesis of colon carcinomas and/or may represent a tumor progression marker (14). The signaling diversity mediated by the type I epidermal growth factor receptor family offers a possible mechanism by which overexpression of the kinase-impaired H-Ryk could bring about cellular transformation The epidermal growth factor receptor tyrosine kinase family extends its signaling diversity by trans-phosphorylation of the kinase impaired c-erbB3, which leads to activation of downstream signaling pathways (26, 27). The kinase-impaired c-erbB3 receptor therefore modulates signals of the type I family of receptors by heterodimerization with c-erbB2/c-erbB4, which are kinase active. The mapping of Ryk receptor to two distinct loci, Ryk-1 and Ryk-2, on human chromosome 3q11–22 and 17p13.3, respectively (28), raises the possibility that there might be a kinase-active partner for the Ryk-1 locus cDNA analyzed in this study. If the Ryk-2 locus does harbor a kinase-active partner, one would speculate that overexpression of the Ryk-1 locus cDNA would result in increased heterodimerization of Ryk-1/Ryk-2 and concomitant chronic activation of downstream signaling pathways, which ultimately give rise to the transformed phenotype. Future efforts will be directed toward delineating the signaling pathway that brings about this transformation.

In conclusion, our findings indicate that H-Ryk is overexpressed in epithelial ovarian cancer, particularly in the more aggressive subtypes. The ability of the NIH3T3:H-Ryk transfectants to form tumors in mice suggests indirectly that mere overexpression of H-Ryk may be important in the pathogenesis of ovarian cancer.

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 is supported by the Imperial Cancer Research Fund. R. M. T. K is a recipient of the Rosina Valerie Howell Scholarship. R. W. is supported by the Helen Tomkinson Award from the British Medical Association.

            
3

The abbreviation used is: RTK, receptor tyrosine kinase.

      
4

Unpublished data.

Fig. 1.

Immunostaining of H-Ryk protein in normal tissues. A and B, small bowel; C and D, breast; E and F, testes; G and H, tonsil. Strong diffuse staining is present in epithelial cells. A, C, E, and G, stained with the H-Ryk 15.2 polyclonal antibody. To verify specificity of the 15.2 polyclonal antibody, immunostaining of the same panel of tissues was carried out in the presence of competing peptide (B, D, F, and H). ×40.

Fig. 1.

Immunostaining of H-Ryk protein in normal tissues. A and B, small bowel; C and D, breast; E and F, testes; G and H, tonsil. Strong diffuse staining is present in epithelial cells. A, C, E, and G, stained with the H-Ryk 15.2 polyclonal antibody. To verify specificity of the 15.2 polyclonal antibody, immunostaining of the same panel of tissues was carried out in the presence of competing peptide (B, D, F, and H). ×40.

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Fig. 2.

Expression analysis of H-Ryk receptor in normal ovary and borderline mucinous tumors. Cellular distribution of the receptor was detected in smooth muscles (A) and stroma (B) of normal ovary. Moderate to high expression of H-Ryk detected in the borderline mucinous tumors (C and D). A–C: ×40; D, ×65.

Fig. 2.

Expression analysis of H-Ryk receptor in normal ovary and borderline mucinous tumors. Cellular distribution of the receptor was detected in smooth muscles (A) and stroma (B) of normal ovary. Moderate to high expression of H-Ryk detected in the borderline mucinous tumors (C and D). A–C: ×40; D, ×65.

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Fig. 3.

Immunostaining of ovarian tumors. Intense signal observed in the epithelial and stromal compartments of serous (A and B), endometrioid (C and D), and clear cell (E and F) tumors. ×40.

Fig. 3.

Immunostaining of ovarian tumors. Intense signal observed in the epithelial and stromal compartments of serous (A and B), endometrioid (C and D), and clear cell (E and F) tumors. ×40.

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Fig. 4.

Analysis of H-Ryk expression in the PEO4 (A) and NIH3T3 transfectants (B). Clarified cell lysates from the respective cell lines were subjected to immunoprecipitation in the absence (−) or presence (+) of competing 15.2 cognate peptide. Arrow, Mr 110,000 band that corresponds to the H-Ryk receptor. In vitro transforming analysis of the PEO4: H-Ryk; ▪, PEO4:H-Ryk 20; ▧, PEO4:H-Ryk 21; , PEO4:H-Ryk 41; □, PEO4:pcDNA3 (C) and NIH3T3:H-Ryk; ▪, NIH3T3:H-Ryk 4; ▧, NIH3T3:H-Ryk13; □, NIH3T3:pcDNA3 (D) as determined by their ability to proliferate indefinitely on soft agar. Cells were cultured at a density of 1 × 104 per 35-mm plate in soft agar containing 10% FCS. Colonies with >50 cells were scored after 3 weeks. Each assay was performed in triplicate on two independent occasions. Nude mice assay of the NIH3T3:H-Ryk 4; – –, NIH3T3:H-Ryk4; ---•---, NIH3T3:H-Ryk4 (+ Matrigel) (E) and; – –, NIH3T3:H-Ryk13; ---•---, NIH3T3:H-Ryk 13 (+ Matrigel) 13 (F) transfectants in the presence or absence of matrigel. Each measurement represents the mean of two independent experiments; bars, SD. A total of 10 mice each were used for the experiments. G–J: Histological assessment of the nude mice tumors. G, fibrosarcoma with irregular area of necrosis (arrow); H, tumor infiltration into the adjacent muscle; I, pleomorphism within tumor cells and their nuclei (arrow, high mitotic index); J, formation of collagen fibers (arrow) as shown by a hematoxylin-van Gieson stain. ×40.

Fig. 4.

Analysis of H-Ryk expression in the PEO4 (A) and NIH3T3 transfectants (B). Clarified cell lysates from the respective cell lines were subjected to immunoprecipitation in the absence (−) or presence (+) of competing 15.2 cognate peptide. Arrow, Mr 110,000 band that corresponds to the H-Ryk receptor. In vitro transforming analysis of the PEO4: H-Ryk; ▪, PEO4:H-Ryk 20; ▧, PEO4:H-Ryk 21; , PEO4:H-Ryk 41; □, PEO4:pcDNA3 (C) and NIH3T3:H-Ryk; ▪, NIH3T3:H-Ryk 4; ▧, NIH3T3:H-Ryk13; □, NIH3T3:pcDNA3 (D) as determined by their ability to proliferate indefinitely on soft agar. Cells were cultured at a density of 1 × 104 per 35-mm plate in soft agar containing 10% FCS. Colonies with >50 cells were scored after 3 weeks. Each assay was performed in triplicate on two independent occasions. Nude mice assay of the NIH3T3:H-Ryk 4; – –, NIH3T3:H-Ryk4; ---•---, NIH3T3:H-Ryk4 (+ Matrigel) (E) and; – –, NIH3T3:H-Ryk13; ---•---, NIH3T3:H-Ryk 13 (+ Matrigel) 13 (F) transfectants in the presence or absence of matrigel. Each measurement represents the mean of two independent experiments; bars, SD. A total of 10 mice each were used for the experiments. G–J: Histological assessment of the nude mice tumors. G, fibrosarcoma with irregular area of necrosis (arrow); H, tumor infiltration into the adjacent muscle; I, pleomorphism within tumor cells and their nuclei (arrow, high mitotic index); J, formation of collagen fibers (arrow) as shown by a hematoxylin-van Gieson stain. ×40.

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Table 1

Expression of H-Ryk in normal tissuesa

OrganImmunoreactivity
Pancreas Islets of Langerhans ++ 
Liver Hepatocytes ++ 
Kidney Tubular cells + 
Small bowel Epithelial cells +++ 
 Vascular smooth muscle ± 
Vas deferens Epithelium + 
 Smooth muscle ++ 
Prostate − 
Uterus Proliferative endometrium − 
 Secretory endometrium + 
 Myometrium ++ 
Fallopian tube Epithelium ++ 
Ovary Vascular smooth muscle ++ 
 Surface epithelium + 
 Stroma + 
Breast Epithelium ++ 
Lung Vascular smooth muscle + 
Heart Myocardium ++ 
 Blood vessels +++ 
Spleen Red pulp ++ 
Lymph node Sinus histiocytes + 
Tonsil Squamous epithelium ++ 
 Lymphoid follicles + 
Skeletal muscle +++ 
Thyroid Epithelial cells ++ 
Brain Blood vessels ++ 
 Ependyma + 
OrganImmunoreactivity
Pancreas Islets of Langerhans ++ 
Liver Hepatocytes ++ 
Kidney Tubular cells + 
Small bowel Epithelial cells +++ 
 Vascular smooth muscle ± 
Vas deferens Epithelium + 
 Smooth muscle ++ 
Prostate − 
Uterus Proliferative endometrium − 
 Secretory endometrium + 
 Myometrium ++ 
Fallopian tube Epithelium ++ 
Ovary Vascular smooth muscle ++ 
 Surface epithelium + 
 Stroma + 
Breast Epithelium ++ 
Lung Vascular smooth muscle + 
Heart Myocardium ++ 
 Blood vessels +++ 
Spleen Red pulp ++ 
Lymph node Sinus histiocytes + 
Tonsil Squamous epithelium ++ 
 Lymphoid follicles + 
Skeletal muscle +++ 
Thyroid Epithelial cells ++ 
Brain Blood vessels ++ 
 Ependyma + 
a

Expression levels are indicated with absent (−), absent to low (±), moderately high (++), and high (+++).

Table 2

Expression of H-Ryk in ovarian tumorsa

Immunoreactivity
SampleHistological typeGrade of differentiationEpitheliumConnective tissueBlood vessels
1b Mucinous cystadenoma − − ++ 
2b Serous cystadenoma − ++ − − 
3c Mucinous − +++ ++ ++ 
4c Mucinous − − ± ++ 
5c Mucinous − ± − − 
6c Mucinous − +++ ++ ++ 
7c Serous − +++ ++ 
8c Serous − ++ ++ ++ 
9c Serous − +++ ++ ++ 
10c Serous − ++ +++ 
11d Serous Moderate +++ ++ ++ 
12d Serous Poor +++ − ++ 
13d Serous Moderate +++ +++ +++ 
14d Serous Poor ± − − 
15d Serous Poor +++ +++ 
16d Serous Poor ++ ++ +++ 
17d Serous Poor ++ − 
18d Serous Moderate 
19d Serous Poor +++ +++ ++ 
20d Mucinous Poor ++ − − 
21d Mucinous Well ± ± 
22d Mucinous Well − 
23d Endometroid Poor ± ± 
24d Endometroid Well ++ ± 
25d Endometroid Moderate ± − − 
26d Endometroid Poor ± 
27d Endometroid Poor ++ − − 
28d Endometroid Poor +++ +++ 
29d Clear cell Poor +++ ++ − 
30d Clear cell Moderate +++ ± ++ 
31d Clear cell Poor +++ 
32d Clear cell Moderate ± − − 
33d Clear cell Moderate ± − − 
Immunoreactivity
SampleHistological typeGrade of differentiationEpitheliumConnective tissueBlood vessels
1b Mucinous cystadenoma − − ++ 
2b Serous cystadenoma − ++ − − 
3c Mucinous − +++ ++ ++ 
4c Mucinous − − ± ++ 
5c Mucinous − ± − − 
6c Mucinous − +++ ++ ++ 
7c Serous − +++ ++ 
8c Serous − ++ ++ ++ 
9c Serous − +++ ++ ++ 
10c Serous − ++ +++ 
11d Serous Moderate +++ ++ ++ 
12d Serous Poor +++ − ++ 
13d Serous Moderate +++ +++ +++ 
14d Serous Poor ± − − 
15d Serous Poor +++ +++ 
16d Serous Poor ++ ++ +++ 
17d Serous Poor ++ − 
18d Serous Moderate 
19d Serous Poor +++ +++ ++ 
20d Mucinous Poor ++ − − 
21d Mucinous Well ± ± 
22d Mucinous Well − 
23d Endometroid Poor ± ± 
24d Endometroid Well ++ ± 
25d Endometroid Moderate ± − − 
26d Endometroid Poor ± 
27d Endometroid Poor ++ − − 
28d Endometroid Poor +++ +++ 
29d Clear cell Poor +++ ++ − 
30d Clear cell Moderate +++ ± ++ 
31d Clear cell Poor +++ 
32d Clear cell Moderate ± − − 
33d Clear cell Moderate ± − − 
a

Immunoreactivity of H-Ryk is indicated with absent (−), minimal (±), low (+), moderate (++), and high (+++).

b

Benign

c

borderline, or

d

malignant tumors of the ovary.

We thank I. Mackenzie and M. Gillmer for providing the ovarian tumors. We are grateful to Sandra Peak and Del Watling at the Imperial Cancer Research Fund animal house for the in vivo experimental work. Helen Mellor is gratefully acknowledged for the color photograph reproductions.

1
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