EGFRvIII was first reported in human glioblastomas. Subsequent reports indicated EGFRvIII protein to be frequently detected in several other human cancers, but not in normal tissues. Our previous studies suggested that EGFRvIII could induce a transformation from ligand-dependent non-tumorigenic cell line to ligand-independent malignant phenotype cells in vitro and in vivo. Transfection of EGFRvIII in MCF-7 cell line resulted in a 3-fold increase in colony formation and significantly enhanced tumorigenicity in nude mice (p < 0.001). EGFRvIII could also induce ErbB-2 phosphorylation. The existence and significance of EGFRvIII transcript in human breast cancer, however, was not reported. In our study, we detected the presence of EGFRvIII mRNA and revealed a high incidence (67. Key words: EGFRvIII; EGFR; LCM; breast cancerDevelopment and progression of breast cancer may result from an accumulation of varied aberrations of genes and their products. Detecting and understanding important molecules will address their roles in carcinogenesis and may be considered to be the prerequisite for the genesis of molecular therapeutics in breast cancer treatment. Overexpression of epidermal growth factor receptor (EGFR) has been detected in a variety of human cancers with 15-90% detection rates, and therefore, it has been thought a possible candidate for cancer therapy. [1][2][3][4][5][6] A relationship between overexpression of EGFR and increased metastatic potential and poor prognosis in breast cancers has been reported. 7,8 EGFRvIII, the most common deletion receptor of EGFRwt, was first reported in human glioblastomas. 9 -11 Subsequently, a few reports demonstrated that EGFRvIII protein was also detected in other human cancers, including breast, ovarian, lung, and medulloblastomas, 12,13 but not in normal tissue. 13,14 In our previous studies, we have observed a high incidence of EGFRvIII protein (62%) in human invasive breast cancer and demonstrated a significantly enhanced tumorigenicity in EGFRvIII transfected MCF-7 breast cancer cells in nude mice. 15,16 Studies to confirm the presence and significance of EGFRvIII transcript and protein in breast cancer, however, are still underdeveloped.To further confirm our early immunohistochemistry results and to ascertain EGFRvIII mRNA expression in human breast cancer tissue to explore the possible role of EGFRvIII in breast cancer genetics, we applied the laser capture microdissection (LCM) combined with RT-PCR detection. The PixCell LCM, a newly developed state-of-the-art technique, allows precise capture of specific cells from tissues comprising mixed cell types. 17,18 LCM can procure pure cell populations for molecular analysis by adhering the selected cells to a thermoplastic film through laser pulse. [17][18][19][20] By applying this technique, we were able to preferentially select "pure" breast cancer cells from mixed cell populations of breast tissues for genetic study. As a result, we detected a high incidence (67.8%) of EGFRvIII transcripts in primary invasive breast can...

Monocyte chemotactic protein (MCP)-

This study was undertaken to determine if acidic or basic fibroblast growth factor (FGF1 or FGF2) or vascular endothelial growth factor (VEGF) alters the radiation response of small bowel after total-body irradiation (TBI). Female C3H mice were treated with various doses of angiogenic growth factor administered intravenously 24 h before or 1 h after TBI. Radiation doses ranged from 7 to 18 Gy. End points measured were the number of crypts in three portions of the small bowel, the frequency of apoptosis of crypt cells at various times after TBI, and the LD50/30 (bone marrow syndrome) and LD50/6 (GI syndrome). Fibroblast growth factors alone, without TBI, decreased the number of crypts per circumference significantly. Among the factors tested, FGF2 caused the greatest decline in baseline crypt number. Despite this decrease in the baseline crypt number, after irradiation the number of surviving crypts was greater in animals treated with growth factor. The greatest radioprotection occurred at intermediate doses of growth factor (6 to 18 pg/mouse). Mice treated with FGF1 and FGF2 had crypt survival curves with a slope that was more shallow than that for saline-treated animals, indicating radiation resistance of crypt stem cells in FGF-treated mice. The LD50/6 was increased by approximately 10% for all treatments with angiogenic growth factors, whether given before or after TBI. Apoptosis of crypt cells was maximum at 4 to 8 h after TBI. The cumulative apoptosis was decreased significantly in animals treated with angiogenic growth factors, and the greatest protection against apoptosis was seen in animals treated with FGF2 prior to TBI. All three angiogenic growth factors tested were radioprotective in small bowel whether given 24 h before or 1 h after irradiation. The mechanism of protection is unlikely to involve proliferation of crypt stem cells, but probably does involve prevention of radiation-induced apoptosis or enhanced repair of DNA damage of crypt cells.

Elevated levels of epidermal growth factor receptor (EGFR) have been detected in a variety of human cancers. Several reports have demonstrated that the Type III EGF receptor deletion-mutant (EGFRvIII) is frequently detected in various human cancers, including breast cancer. We generated and characterized monoclonal antibody against EGFRvIII. We demonstrated that 29% of DCIS, 40% of primary invasive breast cancers and 54% of metastatic lymph nodes express EGFRvIII by immunohistochemical analysis with two monoclonal antibodies. High levels of EGFRvIII expression were detected in about 5% of primary breast cancer and 27% of metastatic lymph-nodes. Furthermore, in the positive samples, the normal mammary gland exhibited negative staining for EGFRvIII, while the tumor cells were positive. The frequency of EGFRvIII expression correlated with breast cancer progression. We also showed that, despite the absence of gene amplification of EGFR in breast carcinoma cells, EGFRvIII was phosphorylated in breast cancer. In addition, approximately 40% of ErbB-2 positive primary breast tumors were found to co-express EGFRvIII. Even more striking is that 75% (3/4) of ErbB-2 positive metastatic lymph node specimens co-expressed with EGFRvIII. Co-expression of EGFRvIII with ErbB-2 in 32D cells amplified downstream signaling cascades and significantly enhanced tumorigenesis in vivo. Furthermore, EGFRvIII mediated constitutively activated and sustained downstream signaling pathways, whereas EGF-ligand induced a transient effect on wt-EGFR-mediated downstream signaling pathways.

Background-Inhibition of GPVI has been proposed as a useful antithrombotic strategy; however, in vivo proof-ofconcept animal studies targeting GPVI are lacking. We evaluated a novel anti-human GPVI monoclonal antibody OM4 Fab in rats. Methods and Results-OM4 Fab specifically inhibited collagen-induced aggregation of rat platelets in vitro with an IC 50 of 20 to 30 g/mL but not ADP and AA-induced platelet aggregation. After intravenous administration of OM4 Fab, a rapid inhibition of ex vivo platelet aggregation was observed with a gradual recovery within 60 to 90 minutes which corresponded to the decline in OM4 Fab plasma concentration and time-dependent decrease in platelet-bound OM4 Fab. In contrast to previous reports in mice, intravenous OM4 Fab did not deplete platelet GPVI. Injection of OM4 IgG caused acute thrombocytopenia. In a modified Folts model of cyclic flow reduction in rat carotid artery, the number of complete occlusions was significantly reduced by intravenous administration of OM4 Fab (20 mg/kg) before or after mechanical injury to the vessel, without prolongation of bleeding time. Conclusion-Fab fragment of the monoclonal antibody OM4 effectively inhibits collagen induced platelet aggregation in vitro and ex vivo, and in vivo thrombosis in rats without prolonging bleeding time. Antibodies against GPVI may have therapeutic potential, inhibiting thrombosis without prolonging bleeding time. (Arterioscler Thromb Vasc Biol.