Expression of activated platelet-derived growth factor receptor in stromal cells of human colon carcinomas is associated with metastatic potential

Platelet-derived growth factor receptor (PDGF-R) expression has been reported in a variety of cancers, including colorectal, breast, lung, ovarian and pancreatic cancers, but the role of PDGF-R expression in the development and progression of colon carcinoma has not yet been elucidated. The purpose of this study was to examine the expression of PDGF and PDGF-R in human colon carcinomas. The expression of PDGF, PDGF-R and phosphorylated PDGF-R (p-PDGF-R) was examined by immunofluorescence in 12 surgical specimens of colon carcinoma and in human colon carcinoma cells growing in the subcutis (ectopic site) and the cecal wall (orthotopic site) of nude mice. In most surgical specimens, tumor cells expressed PDGF-A and -B subunits, without corresponding levels of PDGF-Ralpha and PDGF-Rbeta. PDGF-Rbeta was predominantly expressed by tumor-associated stromal cells and pericytes of tumor vasculature. The expression of PDGF-Rbeta in the stroma was associated with advanced stage disease. Under culture conditions, human colon carcinoma cell lines expressed PDGF-A and -B, but not PDGF-R. In orthotopic tumors, the KM12 cells (Duke's stage B) expressed PDGF-A and -B, but PDGF-Rbeta was expressed only by stromal cells and pericytes in the tumor vasculature. This expression of PDGF-Rbeta by stromal cells and pericytes was higher in tumors growing at the orthotopic site than in those at the ectopic site. The expression of PDGF-Rbeta in the stroma was higher in highly metastatic KM12SM tumors than in low metastatic KM12C tumors. In conclusion, the expression of PDGF-Rbeta in stromal cells is influenced by the organ-specific microenvironment and is associated with metastatic potential.     

Expression of connective tissue growth factor in cartilaginous tumors

BACKGROUND: Connective tissue growth factor (CTGF) predominantly is expressed in hypertrophic chondrocytes and its specific receptors are demonstrated on chondrocytic cells. Therefore, CTGF may be involved in the proliferation and/or differentiation of cartilage cells. In the current study, CTGF expression was examined both in chondrosarcoma and enchondroma to clarify the relation between the expression of CTGF and the grade of malignancy. METHODS: The expression of CTGF and proliferating cell nuclear antigen (PCNA) were analyzed immunohistochemically in 34 cartilaginous tumor specimens. Eighteen tumors were determined to be chondrosarcoma including 8 Grade 1 tumors, 6 Grade 2 tumors, and 4 Grade 3 tumors. The percentage of CTGF positive and PCNA positive cells was quantified using at least 500 cells. RESULTS: CTGF was expressed in 70.1% of enchondroma cells, 84.0% of Grade 1 chondrosarcoma cells, 53.7% of Grade 2 tumor cells, and 26.8% of Grade 3 tumor cells (rho = -0.501; P = 0.0053). In chondrosarcoma cases, CTGF expression was correlated closely with tumor grade (rho = -0.920; P = 0.0001). There was a strong correlation between PCNA expression and tumor grade (rho = 0.907; P < 0.0001) and a strong negative correlation between CTGF and PCNA expression (rho = -0.493; P = 0.0061). In chondrosarcoma cases, patients with high expression of CTGF (>/= 30%) showed higher overall survival compared with those with low expression (< 30%) (P = 0.004). CONCLUSIONS: The current study revealed a correlation between the histologic grade of chondrosarcoma and prognosis, and the concomitant association between CTGF immunostaining and tumor grade and prognosis. Therefore, immunohistochemical staining with CTGF is a useful procedure for assessing the tumor grade and clinical course in patients with chondrosarcoma.     

Expression of PDGF beta-receptors in human meningioma cells.

Meningioma is a generally benign tumor derived from arachnoid tissue. We have investigated the presence of functionally active PDGF-receptors on human meningioma cells in culture. Tumor samples were obtained from 3 surgically removed benign meningiomas and normal arachnoid tissue from an autopsy case. Binding studies were performed by using 125I-labelled recombinant PDGF-AA and PDGF-BB. Only 125I-PDGF-BB showed specific binding to all tumor-cell cultures after incubation of cells for 2 hr at 4 degrees C. Effects of PDGF-AA and PDGF-BB on DNA synthesis were measured as 3H-thymidine incorporation during 48 hr of labelling cells maintained in Eagle's minimum essential medium 0.5% fetal calf serum. PDGF-BB but not PDGF-AA stimulated DNA synthesis in all 3 tumor-cell cultures. Total cellular RNA was analyzed by Northern blotting and hybridization with a 32P-labelled human PDGF beta-receptor probe, and PDGF beta-receptor mRNA was found in both tumor and arachnoid cell cultures. Furthermore, PDGF beta-receptor mRNA was shown to be present in 2 meningioma biopsies and immunohistochemical staining revealed that PDGF beta-receptors are present in meningioma and arachnoid tissues in vivo. It appears that a possible way of maintaining human meningioma cell growth in vivo is through activation of PDGF beta-receptors.     

In situ distribution of the beta-subunit of platelet-derived growth factor receptor in nonneoplastic tissue and in soft tissue tumors

The distribution of the beta-subunit of platelet-derived growth factor receptor (PDGFR-beta) was assessed by a sensitive immunoalkaline phosphatase technique using the monoclonal antibody PR7212. Frozen tissue sections of several nonneoplastic human tissues were stained along with 42 soft tissue sarcomas, 16 benign soft tissue proliferations, and 7 epithelial tumors. In all nonneoplastic tissue, there was intense labeling of cell processes of perivascular fibroblasts or pericytes in and about the walls of muscular blood vessels and of fibroblast cell processes around some glandular and ductal epithelia. No PDGFR-beta was found in the endothelial cells of muscular arteries and veins, but cells of uncertain identity within some capillaries were immunoreactive and the possibility that endothelial cells of some small capillaries express PDGFR-beta could not be excluded. In kidney there was strong labeling of glomerular mesangial cells and interstitial fibroblasts. Some histological types of soft tissue sarcomas were uniformly and strongly labeled with anti-PDGFR-beta, but other types were infrequently labeled or unreactive. The order of decreasing frequency and strength of labeling of the various types of benign and malignant soft tissue proliferations was as follows: benign fibromatosis and neurofibroma greater than malignant fibrous histiocytoma greater than liposarcoma greater than leiomyosarcoma greater than rhabdomyosarcoma. No tumor cell labeling was detected in epithelioid, synovial or clear cell sarcomas, leiomyomas, or carcinomas, but there was usually strong labeling of fibroblast and/or pericyte cell processes within tumor, especially around blood vessels. We conclude that PDGFR-beta is strongly expressed by vascular and stromal tissues of most tumors and normal organs and by tumor cells of several types of soft tissue tumors and proliferations, most notably those of fibroblastic origin.     

Expression of epidermal growth factor receptors in human lung tumors

Using the adjacent histologically normal tissues obtained from the same patients as controls, six human lung tumors were studied for the activities of epidermal growth factor (EGF) receptor binding, and receptor autophosphorylation. There was a 1.2- to 2.8-fold increase in EGF receptor activities in lung tumors due to an increase in the number of receptors without changes in their affinity. The increase had no direct correlation with the degree of differentiation or the type of lung tumors. The elevated expression of EGF receptor may be one of the characteristics in lung tumors. Epidermal growth factor and its receptor also may play a role in the regulatory mechanisms during tumorigenesis.     

Expression of epidermal growth factor receptors in human brain tumors

The expression of receptors for epidermal growth factor (EGF-R) was determined in 29 samples of brain tumors from 22 patients. Primary gliogenous tumors, of various degrees of cancer, five meningiomas, and two neuroblastomas were examined. Tissue samples were frozen in liquid nitrogen immediately after the operation and stored at -70 degrees until use. Cerebral tissue samples from 11 patients who died from diseases not related to the central nervous system served as controls. Immunoprecipitation of functional EGF-R-kinase complexes revealed high levels of EGF-R in all of the brain tumors of nonneuronal origin that were examined. The level of EGF-R varied between tumors from different patients and also between specimens prelevated from different areas of the same tumor. In contrast, the levels of EGF-R from control specimens were invariably low. The biochemical properties of EGF-R in brain tumor specimens were found to be indistinguishable from those of the well-characterized EGF-R from the A-431 cell line, derived from human epidermoid carcinomas. Human brain EGF-R displays a molecular weight of 170,000 by polyacrylamide-sodium dodecyl sulfate gel electrophoresis. It is phosphorylated mainly in tyrosine residues and shows a 2-dimensional phosphopeptide map similar to that obtained with the phosphorylated EGF-R from membranes of A-431 cells. Our observations suggest that induction of EGF-R expression may accompany the malignant transformation of human brain cells of nonneuronal origin.     

Expression of platelet-derived growth factor and its receptors in neuroendocrine tumors of the digestive system.

Carcinoid tumors are slowly growing neuroendocrine neoplasms which often present pronounced fibrosis around the tumor cells. We have previously shown by immunohistochemistry that carcinoid tumors express platelet-derived growth factor (PDGF) beta-receptors on surrounding stromal cells. In this report, 22 midgut carcinoids and 5 endocrine pancreatic tumors were examined for the presence of PDGF with a monoclonal antibody raised against a peptide corresponding to a part of the B-chain of PDGF which reacts strongly with the B-chain and weakly with the A-chain. They were also examined for PDGF alpha-receptors with an affinity-purified polyclonal peptide antibody and for PDGF beta-receptor with the monoclonal antibody PDGFR-B2. PDGF was expressed on tumor cells and on adjacent stroma. PDGF alpha-receptor was seen on clusters of tumor cells and occasionally on adjacent stroma, whereas beta-receptors were seen only in the stroma. Tissue sections from some of these midgut carcinoids were also investigated by in situ hybridization for mRNA of PDGF A- and B-chains as well as alpha- and beta-receptors. By in situ hybridization, abundant expression of mRNA for PDGF beta-receptor and PDGF A-chain was observed in stromal cells adjacent to carcinoid tumor cell clusters, but the mRNA expression in the tumor cells themselves was at a low level. A few clustered tumor cells and stromal cells expressed mRNA for the PDGF alpha-receptor, thus consolidating the immunohistochemical findings. mRNA for the PDGF B-chain was detected in both tumor cells and stroma, but only at low levels. Our data suggest that PDGF is involved in the growth stimulation of the carcinoid tumor cells in an autocrine fashion and in the stimulation of stromal cell growth through paracrine and possibly autocrine mechanisms. Moreover, remarkably strong immunostaining of PDGF and the PDGF alpha-receptor was seen on peripheral nerve fibers.     

Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors.

Analysis of genomic organization and expression of platelet-derived growth factor receptors (PDGFR) and epidermal growth factor receptor (EGFR) in human malignant gliomas showed amplification and overexpression of both receptors in distinct subsets of tumors. Amplification of the alpha PDGFR was detected in 4 of 50 glioblastomas (8%). EGFR was amplified in 9 of the 50 tumors (18%). Western blot analysis showed elevated expression of alpha PDGFR and EGFR proteins in 4 (24%) and 3 (18%), respectively, of 17 tumor specimens analyzed. Increased production of alpha PDGFR as well as EGFR proteins was observed in the presence or absence of gene amplification. Three of the 4 tumors with elevated levels of alpha PDGFR also overexpressed the beta PDGFR, which was present as a single copy gene in all 50 tumors analyzed. Our findings suggest that the amplification and/or overexpression either of EGFR or of the alpha PDGFR along with the coordinate overexpression of the beta PDGFR can contribute to the malignant phenotype of distinct subsets of human glioblastoma.     

Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human gallbladder lesions

The aim of this study was to investigate the expression of platelet-derived endothelial growth factor (PD-ECGF) in human gallbladder carcinomas to elucidate its role in angiogenesis and tumour progression. To this end, 56 archival surgical specimens of gallbladder lesions were examined for PD-ECGF/thymidine phosphorylase (TP) expression by immunohistochemistry and the PD-ECGF/TP protein level was assessed in five fresh specimens of gallbladder carcinoma by enzyme-linked immunosorbent assay (ELISA). Hyperplastic epithelial cells and adenoma cells showed no or faint staining with PD-ECGF/TP. Out of 43 gallbladder carcinomas, 27 (63%) showed moderate to strong immunoreactivity in the cytoplasm and nuclei of the tumour cells. PD-ECGF/TP immunoreactivity in stromal infiltrating cells was detected in 43% (3/7) hyperplasias, 17% (1/6) adenomas and 86% (37/43) carcinomas. PD-ECGF/TP protein levels in carcinoma tissues were higher than those in corresponding normal mucosa. PD-ECGF/TP expression did not correlate with angiogenesis, but significantly correlated with depth of invasion, lymph node metastasis, and tumour stage. These results overall suggest that PD-ECGF/TP produced by both cancer cells and infiltrating cells is associated with tumour progression in human gallbladder carcinoma.     

Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase in human breast cancer

We have investigated the expression of platelet-derived endothelial-cell growth factor/thymidine phosphorylase (PD-ECGF/dThdPase) in human breast-cancer tissues by the immunocytochemical method using anti-PD-ECGF/dThdPase monoclonal antibody. Out of 100 invasive-ductal-carcinoma tissue samples, 39 (39%) were evaluated as PD-ECGF/dThdPase-positive. The expression of PD-ECGF/dThdPase was identified mainly in the cytoplasma of tumor cells. The expression of PD-ECGF/dThdPase was significantly associated with the microvessel density assessed by immunostaining to factor-VIII-related-antigen (p < 0.05). However, there was no correlation between expression of PD-ECGF/dThdPase and menopausal status, tumor size, axillary lymph-node metastases, hormone-receptor status, epidermal-growth-factor receptor, or erb-B-2-protein and p53-protein expression. We suggest that expression of PD-ECGF/dThdPase plays an important role in the promotion of angiogenesis in human breast cancer. © 1995 Wiley-Liss, Inc.     

Evidence for functional platelet-derived growth factor receptors on MG-63 human osteosarcoma cells

The specific interaction of platelet-derived growth factor (PDGF) with the human osteosarcoma cell line MG-63 was studied. Scatchard analysis of 125I-PDGF binding to MG-63 cells indicated there were 32,000 specific PDGF-binding sites per cell with a Kd of 2.4 X 10(-11) M. Unlabeled PDGF blocked the specific binding of labeled PDGF to MG-63 cells at concentrations greater than 1 ng/ml. When assayed for phosphorylation of MG-63 membrane vesicles, PDGF was shown to stimulate a dose-dependent phosphorylation of a protein (phosphoprotein with a molecular weight of 185,000) which was stable in 1 M NaOH. In the absence of PDGF, a prominent alkali-stable phosphoprotein with a molecular weight of 116,000 was noted. PDGF also stimulated a dose-dependent increase in [3H]aminoisobutyric acid uptake, [3H]thymidine incorporation, and cell proliferation. When tested for secretion of PDGF-like factors, the mitogenic activity of MG-63-conditioned serum-free medium was not blocked by anti-PDGF antiserum. Concentrated MG-63-conditioned medium did not compete with 125I-PDGF for specific receptor sites on diploid fibroblasts. Therefore, MG-63 osteosarcoma cells have functional PDGF receptors and do not secrete PDGF-like mitogens.     

Pediatric KIT wild-type and platelet-derived growth factor receptor alpha-wild-type gastrointestinal stromal tumors share KIT activation but not mechanisms of genetic progression with adult gastrointestinal stromal tumors

Fewer than 15% of gastrointestinal stromal tumors (GIST) in pediatric patients harbor KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations in contrast to a mutation rate of 80% in adult GISTs. However, some therapeutic inhibitors of KIT have efficacy in pediatric GIST, suggesting that KIT may, nevertheless, play an important role in oncogenesis. In adult GIST, characteristic cytogenetic changes occur during progression to malignancy. A better understanding of mechanisms of genetic progression and KIT and PDGFRA transforming roles in pediatric GIST might facilitate treatment advances. KIT and PDGFRA mutation analysis was done in 27 pediatric GISTs. The activation status of KIT, PDGFRA, and downstream signaling intermediates was defined, and chromosomal aberrations were determined by single nucleotide polymorphism assays. Mutations in KIT or PDGFRA were identified in 11% of pediatric GISTs. KIT and the signaling intermediates AKT and mitogen-activated protein kinase were activated in pediatric GISTs. In particular, most pediatric KIT-wild-type GISTs displayed levels of KIT activation similar to levels in adult KIT-mutant GISTs. Pediatric KIT-wild-type GISTs lacked the typical cytogenetic deletions seen in adult KIT-mutant GISTs. Notably, most pediatric KIT-wild-type GISTs progress to malignancy without acquiring large-scale chromosomal aberrations, which is a phenomenon not reported previously in malignant solid tumors. KIT activation levels in pediatric KIT-wild-type GISTs are comparable with those in KIT-mutant GISTs. Therapies that inhibit KIT activation, or crucial KIT signaling intermediates, should be explored in pediatric KIT-wild-type GIST.     

Expression of platelet-derived endothelial cell growth factor in prostatic adenocarcinoma

Angiogenesis is thought to play critical roles in local tumor growth and eventual metastasis. No studies have examined the expression of platelet-derived endothelial cell growth factor (PD-ECGF) in prostatic tissues. Prostatic tissues were obtained from 36 prostatic adenocarcinoma patients. We assessed the expression of PD-ECGF using ELISA and immunohistochemistry. The mean level of PD-ECGF in prostatic adenocarcinomas was higher than that in neighboring normal prostatic tissues in ELISA. Immunohistochemistry showed that the expressions of PD-ECGF, which were associated with increase of microvessel count, were found in the endothelial cells, macrophages, lymphocytes, or fibroblasts. These results suggest that PD-ECGF is involved in the development of prostatic adenocarcinoma.     

Correlation of KIT and platelet-derived growth factor receptor alpha mutations with gene activation and expression profiles in gastrointestinal stromal tumors

Activating mutations of KIT and platelet-derived growth factor receptor alpha (PDGFRA) are known to be alternative and mutually exclusive genetic events in the development of gastrointestinal stromal tumors (GISTs). We examined the effect of the mutations of these two genes on the gene expression profile of 22 GISTs using the oligonucleotide microarray. Mutations of KIT and PDGFRA were found in 17 cases and three cases, respectively. The remaining two cases had no detectable mutations in either gene. The mutation status of KIT and PDGFRA was directly related to the expression levels of activated KIT and PDGFRA, and was also related to the different expression levels of activated proteins that play key roles in the downstream of the receptor tyrosine kinase III family. To evaluate the impact of mutation status and the importance of the type of mutation in gene expression and clinical features, microarray-derived data from 22 GISTs were interpreted using a principal component analysis (PCA). Three relevant principal component representing mutation of KIT, PDGFRA and chromosome 14q deletion were identified from the interpretation of the oligonucleotide microarray data with PCA. After supervised analysis, there was at least a two fold difference in expression between GISTs with KIT and PDGFRA mutation in 70 genes. Our findings demonstrate that mutations of KIT and PDGFRA affect differential activation and expression of some genes, and can be used for the molecular classification of GISTs.     

Expression and functional analysis of platelet-derived growth factor in uterine leiomyomata

OBJECTIVE: To examine the expression of PDGF and its receptors in leiomyoma tissue and to investigate the regulation of PDGF on leiomyoma cell proliferation. MATERIALS AND METHODS: The expression of PDGF and its receptors was examined in 21 pairs of uterine leiomyoma and the adjacent myometrium tissues using an immunostaining method. Paired cultures of leiomyoma and normal myometrium cells were established and treated with PDGF. Total RNA was extracted from leiomyoma and myometrial cells for detection of the expression of proliferating cell nuclear antigen (PCNA) and collagen alpha1 (I, III) by semi-quantitative PCR. RESULT: The expression of PDGF-AA and PDGF-BB in leiomyoma tissue is obviously higher than that in the matched myometrial tissue (P < 0.05). In addition, the expression of PDGF-BB in secretory phase is higher than that in proliferative phase of the menstrual cycle (P = 0.027) in leiomyoma tissue. The expression of PCNA and collagen alpha1 (I) were increased in both leiomyoma and myometrial cells after treatment of PDGF, whereas the expression levels were greater in leiomyoma cells than that in myometrial cells. CONCLUSION: The expression of PDGF and its receptors in leiomyoma and myometrial tissue varied during the menstrual cycle. PDGF may play a role in the pathogenesis of leiomyomas through a mechanism involved in not only the proliferation of leiomyoma cells but also excessive expression of extra cellular molecules.     

Effects of interferon on tumor tissue content in liver metastases of human carcinoid tumors

In 21 patients ultrasound-guided cutting biopsies, from carcinoid metastases of the liver, were taken before and after therapy with alpha-interferon. Each biopsy was examined under light microscopy and the amount of tumor tissue and connective tissue was quantified and then correlated to objective response to interferon therapy. A significant reduction of the amount of tumor tissue, in spite of unaltered metastatic size and a corresponding increase in connective tissue, was seen after interferon therapy. A more pronounced reduction of tumor tissue occurred after long-term interferon therapy. A positive correlation between objective therapy response and tumor tissue reduction was also present. Patients responding poorly, or not at all, to therapy did not show any significant decrease in tumor tissue. Since treatment with immune response modifiers is expected to increase in the near future, it is important to choose the right investigations for therapy monitoring, and since all patients in this investigation had unchanged tumor size on repeated radiological examinations, it is obvious that microscopic examination of core biopsies is a better method for evaluating effects of long-term therapy than tumor size measurement with radiological techniques. Further, the results may indicate that interferon exerts a cytotoxic effect on carcinoid tumor cells in vivo.     

Expression of messenger RNAs for platelet-derived growth factor and its receptors in human sarcoma cell lines

Growth factors of the platelet-derived growth factor (PDGF) family have been thought to possess autocrine functions in certain neoplasms of mesenchymal and glial origin. This notion has been based on observations that these tumors express PDGF genes and produce PDGF-like growth factors. Corresponding data on PDGF receptor expression in sarcoma cell lines is essentially lacking. The cloning of cDNA for 2 distinct PDGF receptors with different abilities to recognize the members of the PDGF family and availability of recombinant PDGF for binding studies have recently made it possible to study the expression of both receptor types in tumor cell lines. We present here a study on 8 human sarcoma cell lines, and show a large variability and independency in the expression of the 2 PDGF receptor types as well as of the genes encoding the corresponding ligands.     

9p21 locus analysis in high-risk gastrointestinal stromal tumors characterized for c-kit and platelet-derived growth factor receptor alpha gene alterations

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are noncomplex sarcomas that often are due to c-kit-activating and platelet-derived growth factor receptor alpha gene (PDGFRalpha)-activating mutations and perturbations of their related signaling pathways. Molecular and cytogenetic findings have indicated correlations between tumor progression and high-risk GISTs with c-kit mutations, the overexpression of genes such as ezrin, and losses at 9p. In particular, it was reported recently that malignant GISTs showed alterations in the p16INK4a gene located at the 9p21 locus. METHODS: To assess the involvement of p14ARF and p15INK4b in addition to p16INK4a in GISTs, the authors undertook a molecular and cytogenetic study of the 9p21 locus. A series of 22 pre-Gleevec era, cryopreserved, high-risk GISTs that were characterized well in terms of KIT and PDGFRalpha receptors were investigated for mRNA expression, homozygous deletions, mutations, and promoter methylation of locus 9p21, in some instances complemented by fluorescent in situ hybridization studies. RESULTS: The results indicated the loss of p16INK4a mRNA expression in 41% of the GISTs, mainly due to the homozygous deletion of both the p16INK4a gene and the p14ARF gene (24%). No mutations were found, and promoter methylation (detected by means of methylation-specific polymerase chain reaction analysis in 27% of tumors) was restricted mainly to the p15INK4b gene (20%). It is noteworthy that, in all of the methylated GISTs, the epigenetic promoter alteration was coupled with mRNA expression. CONCLUSIONS: Alterations in the 9p21 locus were found cumulatively in 54% of the tumors in the current series and were represented mainly by the loss of tumor suppressor gene expression. The p16INK4a deletion, which always was coupled with p14ARF gene loss, seemed to be the most common 9p21 inactivation mechanism.