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(Objectives) We herein assessed the influence of Epidermal Growth Factor Receptor (EGFR) gene mutations on EGFR expression levels, downstream mediators such as Akt or ERK, and overall survival in patients with ovarian cancer. Study Design) EGFR mutation status was analyzed by direct sequencing in 102 Japanese ovarian cancer patients. The EGFR expression, phosphorylated Akt (pAkt), and phosphorylated ERK (pERK) were determined by immunohistochemistry. (Results) Twenty-nine EGFR gene mutations were detected in 24 of 102 patinets (23.5%). EGFR mutations were observed in 27.9% (19/68) in serous adenocarcinomas, 15.0% (3/20) in clear cell adenocarcinomas, and 66.7% (2/3) in mucinous adenocarcinomas, while no mutations were observed in endometrioid adenocarcinomas (0/11). Protein expression of EGFR, pAkt, and pERK were detected in 47 (46.1%), 49 (48%), and 17 (16.7%) of patients, respectively. EGFR gene mutations, EGFR and pERK expression were not associated with a poor prognosis. In a multivariate analysis, a High pAkt expression was found to be a significant predictor for both the progression free survival (p=0.017) and overall survival (P=0.025). (Conclusion) EGFR gene mutations were frequently observed in not only non-small-cell lung cancer (NSCLC), but also in ovarian cancer in Japanese patients. the selective EGFR inhibitor Gefitinib might therefore offer some benefit in patients with EGFR mutations in ovarian cancer. Our results indicate that the Akt, but not necessarily EGFR, is one of the most important target in the response of the platinum-based chemotherapy and prognosis for ovarian cancer patients.

Growth of solid tumors depends on angiogenesis, the process by which new blood vessels develop from the endothelium of a pre-existing vasculature. Tumors promote angiogenesis by secreting various angiogeneic factors, and newly formed blood vessels induce tumor cell proliferation and invasiveness. Ovarian carcinomas have a poor prognosis, often associated with multifocal intraperitoneal dissemination accompanied by intense neovascularization. The degree of angiogenesis of ovarian carcinomas may directly influence the clinical course of the disease. Although a growing body of evidence indicates that angiogenic intensity may play a prognostic role in gynecological malignancies including ovarian carcinomas, the related biological mechanisms remain to be further elucidated. In this review, we describe current knowledge pertaining to mechanisms and regulation of angiogenesis in ovarian carcinomas with special reference to our recent research results.

Cell transplantation and regeneration therapy are potentially new therapeutic approaches for cardiovascular disease. Transgenic (tg) animals for reporter genes would be useful to follow the cell lineage and differentiation during development and regeneration processes. In the present study, we developed green fluorescent protein (GFP)-tg rats and evaluated them as a tool for the study of cardiomyocyte transplantation and regeneration. The myocardium and bone marrow cells derived from GFP-tg rats strongly expressed GFP. Because neonatal rat cultured cardiomyocytes also strongly expressed GFP, we transplanted GFP-tg rat-derived cardiomyocytes in a rat myocardial infarction (MI) model. Survival of GFP-tg rat-derived cardiomyocytes was confirmed. We further investigated whether bone marrow cells could differentiate into cardiomyocytes using this GFP-tg rat-derived bone marrow cells in vitroand in vivo. GFP-tg rat-derived bone marrow cells differentiated into cardiomyocyte- like cells (cardiac troponin I-expressed cells) by co-culture with wild rat cultured cardiomyocytes in vitro. Furthermore, differentiation of bone marrow cells into cardiomyocyte-like cells was observed by injection of GFP-tg rat-derived bone marrow cells in a rat MI model in vivo. These findings suggest that GFP-tg rats are a useful and valuable tool for the study of transplantation and regeneration in myocardium.

An association between deregulation of homeobox (HOX) gene expression and oncogenic transformation has been recently reported in human tumors. In this study, we investigated HOXgene expression and mutation in cervical carcinoma cells. Using reverse transcription‐PCR, 11 human cervical carcinoma cell lines and 14 normal cervical tissue samples were examined for mRNA expression of the 39 class I HOXgenes. DNA samples from 11 cell lines were tested for mutations in exons 1 and 2 of the HOXA10and A13genes using overlapping primer pairs which also cover intron 1 of these genes. HOXA1, B2, B4, C5, C10and D13genes were expressed in 8, 7, 9, 9, 9 and 11 of 11 cervical carcinoma cell lines, respectively, but not in any of the normal cervical tissues. HOXA9, A11, A13, B5, C4, D3and D9genes were expressed in all cell lines and normal tissues. In contrast, 13 of 39 HOXgenes were silent in all materials examined. Single‐strand conforma‐tional polymorphism and sequence analysis revealed a C insertion after base 1042 and/or a G to C substitution at base 1113 in intron 1 of the HOXA13gene in 4 of 11 cell lines, however, neither deletions nor mutations were detected in exons 1 and 2 of the HOXA10and A13genes. Our data suggest that the expression of HOXA1, B2, B4, C5, C10and D13genes might be involved in the process leading to the transformation of normal cervical cells. (Cancer Sci 2003; 94: 437–441)

The objective of the present study was to produce rat offspring by intracytoplasmic sperm injection (ICSI) using a Piezo-driven micromanipulator. Transgenic male rats carrying a green fluorescent protein gene (GFP: homozygous) were used as sperm donors. The epididymal spermatozoa were suspended and sonicated in m-KRB medium and were frozen in the same medium at −20°C until use. When the sperm heads were aspirated into injection pipettes 7–10 μm in diameter and introduced into oocytes from the Wistar strain, no offspring resulted from the transfer of 59 eggs. In contrast, the sperm heads were hung on the tip of injection pipettes 2–4 μm in diameter and introduced into the oocytes, use of Piezo resulting in the production of 18 transgenic offspring carrying the GFP gene from 181 eggs transferred. The oocytes from the Sprague–Dawley strain also supported full-term development following ICSI with three offspring resulting from 163 transferred eggs. In an additional ICSI trial, spermatozoa from infertile transgenic rats carrying human lactalbumin with the thymidine kinase gene (LAC3: heterozygous) were used. The spermatozoa of the LAC3 transgenic rats appeared to be defective and immotile because of the expression of thymidine kinase in the testes, and no ICSI offspring resulted from 218 transferred eggs. These results suggest that ICSI is applicable in rats when Piezo-driven smaller pipettes are used to inject sperm heads together with a limited amount of the surrounding medium and that the ability of isolated sperm heads to participate in normal embryo development is maintained under the cryopreservation conditions employed.

Biopharmaceuticals intended for humans are immunogenic in animals. Antibodies associated with their administration make it difficult to perform repeated-dose pharmacology and toxicology studies in animals. Despite suggestions to solve this problem with transgenic animal technology, an effective strategy has not yet been reported. The objective of the present study was to provide an efficient strategy to develop rats tolerant to biopharmaceuticals such as human gene-based proteins. The present study used transgenic rat lines (lines 311-6, 308-5, and 305-1) carrying a fusion gene designed to express the human growth hormone (hGH) gene under the control of the bovine αS1 casein gene promoter. Three lactating females with the transgene, produced approximately 4 mg/ml, 300 μg/ml, and 10 ng/ml in their milk. Male 8-week-old rats from these three lines were immunized with hGH three times (week 0, 1, and 3 ) and the production of antibodies against hGH in their sera were examined at week 4. While the hGH serum antibody titers increased over 1000-fold in wild-type control rats, there was no detectable antibody against hGH in the sera of these three transgenic lines. Human growth hormone in their sera was undetectable (lines 308-5 and 305-1) or much lower than the endogenous biologic level of rat growth hormone (line 311-6). Importantly, lines 308-5 and 305-1 developed tolerance to hGH without detectable hGH in their sera and these lines will be very useful for the repeated dose pharmacology and toxicology studies. These results suggest that a milk protein promoter can be a useful tool to develop transgenic rats that are tolerant to biopharmaceuticals intended for humans.

The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT11) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.

Objective.The correlation between the gene expression of various angiogenic factors and in vitroinvasive activity in 16 human gynecological cancer cell lines was investigated.

The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37°C for a period of 48hr. Cells were subsequently cultured at 33°C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1∼9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33°C with a doubling time of 19–21hr. In contrast, cells did not grow at 37 and 39°C due to the reduced expression of large T-antigen, supporting temperature-dependent cell growth. TR-iBRBs expressed GLUT1 and exhibited 3- O-methyl- D-glucose (3-OMG) uptake activity. This 3-OMG uptake was saturable with a Michaelis–Menten constant of 5.56±0.51m Mand a maximum uptake rate of 45.3±2.6nmolmin−1mg protein−1. P-Glycoprotein, with a molecular weight of ∼180KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1bandmdr 2were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells.

Purpose. To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). Methods. Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37°C during the first period of 3 days. Cells were subsequently cultured at 33°C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. Results. Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1∼5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33°C with a doubling-time of 35∼40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by α-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was fivefold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. Conclusions. Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.

Purpose. Postneonatal neovascularization is thought to result exclusively from the proliferation, migration, and remodeling of fully differentiated endothelial cells (ECs). Recently, it has been reported that bone marrow contains cells which can differentiate into ECs and contribute to neoangiogenesis in adult species. In this study, we tried to establish conditionally immortalized endothelial cell lines (TR-BME) derived from rat bone marrow. Methods. Mononuclear cells were isolated and differentiated into ECs at 37°C from the bone marrow of a transgenic rat harboring temperature-sensitive SV40 large T-antigen (ts T-Ag) gene. Then, the cells were transferred and incubated at 33°C, a permissive temperature for ts T-Ag. Expression of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1, 2, Tie-1, 2 and von Willebrand factor (VWF) were assayed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR). Results. We have established three cell lines incorporating 1,1′-dioctadecyl-3,3,3′,3-tetramethylindo-carbocyanine perchlorate (DiI-Ac-LDL) with a spindle shape. One of these, clone 2, strongly expressed VEGFR-2, and weakly expressed VEGFR-1 and VWF. In contrast, clone 8 showed strong expression of Tie-1, 2, and VWF, and weak expression of VEGFR-1,2. All markers were expressed strongly in clone 3. Conclusions. These data confirm that the above three TR-BME cells are novel ECs derived from bone marrow progenitors.

Although it is well established that estrogen deficiency causes osteoporosis among the postmenopausal women, the involvement of estrogen receptor (ER) in its pathogenesis still remains uncertain. In the present study, we have generated rats harboring a dominant negative ERα, which inhibits the actions of not only ERα but also recently identified ERβ. Contrary to our expectation, the bone mineral density (BMD) of the resulting transgenic female rats was maintained at the same level with that of the wild-type littermates when sham-operated. In addition, ovariectomy-induced bone loss was observed almost equally in both groups. Strikingly, however, the BMD of the transgenic female rats, after ovariectomized, remained decreased even if 17β-estradiol (E2) was administrated, whereas, in contrast, the decrease of littermate BMD was completely prevented by E2. Moreover, bone histomorphometrical analysis of ovariectomized transgenic rats revealed that the higher rates of bone turnover still remained after treatment with E2. These results demonstrate that the prevention from the ovariectomy-induced bone loss by estrogen is mediated by ER pathways and that the maintenance of BMD before ovariectomy might be compensated by other mechanisms distinct from ERα and ERβ pathways.

Human urinary erythropoietin has been highly purified by a combination of conventional purification methods and immunoadsorbent columns packed with hybridoma-produced antibodies against contaminants that seemed difficult to separate from erythropoietin by the usual means. By using the partially purified erythropoietin as an antigen, three hybridoma clones have been obtained that secrete monoclonal antibodies against erythropoietin. One of the clones has been quite stable, with a rapid growth rate and high production of antibody. Western blotting technique with monoclonal antibodies revealed occurrence of two species of erythropoietin. The monoclonal antibody will be useful as a probe for the purification of erythropoietin and for further studies of the hormone and its mechanism of action.

Erythropoietin (EPO), a heavily glycosylated protein, is a major stimulatory factor in erythropoiesis. The human EPO gene was engineered for expression in animal cells. Recombinant EPOs produced in two kinds of cells were isolated and their properties compared with those of human urinary EPO. The results indicated that the carbohydrates attached to the EPO peptide are responsible for the different biological activities of these proteins. The biological activities of recombinant glycoproteins produced in heterologous systems may vary depending on the host cells in which the proteins are modified.

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol−1of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivobioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol−1of EPO, there was no further increase in their activity over 12.4 mol mol−1of EPO. On the other hand, an inverse relationship between thein vitrobioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivobioactivity of some fractions with low sialic acid contents was increased after treatment with α2,6-sialyltransferase, but thein vivobioactivity of the other fractions with high sialic acid contents was either decreased or not affected.

We have established four hybridoma cells that produce monoclonal antibodies (MoAbs) R2, R4, R6, and R12 directed toward recombinant human erythropoietin (rHuEPO). MoAbs R2, R4, and R6 bound to EPO with high affinities (kd = ∼2, 4, and 1 nmol/L, respectively) but MoAb R12 had a low affinity (240 nmol/L). These antibodies inhibited the biological activity of rHuEPO and EPOs from humans, rats, mice, and rabbits. This inhibition was due to the blocking of EPO binding to the target cells. The fully deglycosylated rHuEPO bound to the MoAbs, indicating that they recognized peptide sequences of the antigen but not the carbohydrates attached to the antigen. An immunosorbent column with the immobilized MoAb R2 was effective for the rapid purification of EPO. MoAb R6 bound to EPO at a site(s) different from those to which other MoAbs bound. Based on this finding, a sensitive and rapid enzyme-linked immunosorbent assay of EPO, in which EPO was sandwiched between two MoAbs (R2 and R6), was developed. The assay measured plasma levels of EPO as low as 5 mU/mL within several hours.