Establishing a laboratory animal model from a transgenic animal: RasH2 mice as a model for carcinogenicity studies in regulatory science.

Transgenic animal models have been used in small numbers in gene function studies in vivo for a period of time, but more recently, the use of a single transgenic animal model has been approved as a second species, 6-month alternative (to the routine 2-year, 2-animal model) used in short-term carcinogenicity studies for generating regulatory application data of new drugs. This article addresses many of the issues associated with the creation and use of one of these transgenic models, the rasH2 mouse, for regulatory science. The discussion includes strategies for mass producing mice with the same stable phenotype, including constructing the transgene, choosing a founder mouse, and controlling both the transgene and background genes; strategies for developing the model for regulatory science, including measurements of carcinogen susceptibility, stability of a large-scale production system, and monitoring for uniform carcinogenicity responses; and finally, efficient use of the transgenic animal model on study. Approximately 20% of mouse carcinogenicity studies for new drug applications in the United States currently use transgenic models, typically the rasH2 mouse. The rasH2 mouse could contribute to animal welfare by reducing the numbers of animals used as well as reducing the cost of carcinogenicity studies. A better understanding of the advantages and disadvantages of the transgenic rasH2 mouse will result in greater and more efficient use of this animal model in the future.

Population-averaged standard template brain atlas for the common marmoset (Callithrix jacchus).

Advanced magnetic resonance (MR) neuroimaging analysis techniques based on voxel-wise statistics, such as voxel-based morphometry (VBM) and functional MRI, are widely applied to cognitive brain research in both human subjects and in non-human primates. Recent developments in imaging have enabled the evaluation of smaller animal models with sufficient spatial resolution. The common marmoset (Callithrix jacchus), a small New World primate species, has been widely used in neuroscience research, to which voxel-wise statistics could be extended with a species-specific brain template. Here, we report, for the first time, a tissue-segmented, population-averaged standard template of the common marmoset brain. This template was created by using anatomical T(1)-weighted images from 22 adult marmosets with a high-resolution isotropic voxel size of (0.2 mm)(3) at 7-Tesla and DARTEL algorithm in SPM8. Whole brain templates are available at International Neuroinformatics Japan Node website, http://brainatlas.brain.riken.jp/marmoset/.

Basic concept of development and practical application of animal models for human diseases.

The "humanized mouse" is a mouse harboring functioning human tissues used as in vivo human models for both physiological and pathological conditions. The NOD/Shi-scid IL2rgamma(null) (NOG) mouse, an excellent immunodeficient mouse used as the basis for the humanized mouse, requires strict genetic and environmental control for production and use in experiments. Genetic control using marker-assisted selection is described. In addition, NOG mice are easily affected by microbiological and proximate environmental factors, which can cause severe damage to the mice in some cases. Therefore, rigorous microbiological and environmental controls are necessary to ensure reproducibility of experimental results. At the end of this chapter, future aspects of the application of "humanized mice" based on novel super-immunodeficient mice such as NOG mice and Rag2(null) IL2rgamma(null) mice in biomedical research and testing are briefly reviewed.

B-raf, a new member of the raf family, is activated by DNA rearrangement.

Complementary DNA clones of a putative transforming gene were isolated from NIH 3T3 cells transformed with human Ewing sarcoma DNA. The gene was termed B-raf because it is related to but distinct from c-raf and A-raf. It appears that substitution in the amino-terminal portion of the normal B-raf protein confers transforming activity to the gene.

Amplification of the structurally and functionally altered epidermal growth factor receptor gene (c-erbB) in human brain tumors.

By using Southern blot analysis, we found that in two cases of human glioblastoma multiforme, cells carried amplified c-erbB genes which bore short deletion mutations within the ligand-binding domain of the epidermal growth factor (EGF) receptor. The products of these mutated c-erbB genes were about 30 kilodalton (kDa) smaller than the normal 170-kDa EGF receptor, and the tumor cell membrane fractions containing the 140-kDa abnormal EGF receptor showed a significant elevation of tyrosine kinase activity without its ligand. In view of the similarity to the activated viral and cellular erbB genes in the avian system, these mutated and overexpressed EGF receptors might play a role in the onset or development of human glioblastoma cells.

Ascites form of a human cancer serially transplantable in nude mice.

Human adenocarcinoma cells injected into the peritoneal cavities of BALB/c nude mice (nu/nu) induced ascites carcinoma. The inoculant was obtained from subcutaneous tumors produced in nude mice by an injection of ascites cells from a patient with carcinomatous peritonitis caused by mucinous adenocarcinoma of the stomach. An ascitic fluid began to accumulate 45 days after inoculation and reached the maximum volume within 120 days. Dispersed stomach cancer cells in the ascites could be serially transplanted in nude mice in an ascites form. The morphology of these cells was similar to that of the original cells in the ascitic fluid of a patient with carcinomatous peritonitis.

Morphology and function of ganglio-N-tetraosylceramide-positive lymphocyte mediators of natural killer activity.

More than 90% of nylon wool-passed spleen cells from nude mice reacted with ganglio-N-tetraosylceramide (asialo GM1) which eliminated mouse natural killer (NK) cell activity in the presence of complement. The successful enrichment of asialo GM1-positive (asialo GM1+) cells made possible the demonstration that the number of asialo GM1+ cells was correlated with NK cell activity as well as with binding frequency to YAC-1 target cells. Morphologically, by light and immunoelectron microscopic examinations, the enriched asialo GM1+ cells were composed of 90% lymphocytes and 10% large cells with the characteristics of monocytes. However, the asialo GM1+ lymphocytes (but not the monocytes) bound to the target cells and gave rise to the asialo GM1+ monocytes were sometimes observed to bind to target cells, cytolytic features of the target cells were not demonstrated. These results strongly suggest that asialo GM1+ lymphocytes are essential for NK cytolysis by directly binding to target cells.

Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.

BACKGROUND: Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells. METHODS: We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice. RESULTS: The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERM5-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo. CONCLUSION: Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

Plasma protein production by human tumors xenotransplanted in nude mice.

For detection of plasma proteins produced by human malignant tumors, a survey of blood plasma obtained from nude mice bearing serially transplanted human tumors was performed by immunoelectrophoresis and the double immunodiffusion technique. Among 34 lines including 18 types of human tumors, human specific plasma proteins were demonstrated in the plasma of nude mice transplanted with two lines of renal cell carcinoma, one adenocarcinoma of the colon, and one squamous cell carcinoma of the maxillary sinus. These tumors can be designated as "ectopic" plasma protein-producing tumors since the organs or tissues from which they originated are not considered to be usual sites of plasma protein synthesis. Plasma protein production, as well as that of alpha1-fetoprotein, was also found in one line of hepatoblasotma and three lines of yolk sac tumors. The above tumors were shown to produce one or more of the following 10 of 20 plasma proteins examined: albumin, prealbumin, alpha1-antitrypsin, ceruloplasmin, alpha2-macroglobulin, hemopexin, haptoglobin, C3 and C4 component of complement, and transferrin. An immunochemical demonstration of human specific cancer products observed in human tumors xenotransplanted into nude mice may provide a new approach for investigating the metabolism of neoplastic cells.

Establishment of different clonal strains from a human sarcoma of the stomach: tumorigenic heterogeneity in athymic nude mice.

Twenty five clonal strains have been isolated from a single human sarcoma of the stomach. Two different types of clones have been recognized by their morphology and behavior in vitro. Type I clones were characterized by criss-crossed arrays and multilayers with high terminal density. Type II clones grew in a well-organized monolayer with lower saturation density. Although both types of clones exhibited fibroblastic appearance, type I clones showed a more rounded, refractile shape. The cells of this type showed multiple regions of criss-crossed arrays and multilayers throughout the culture vessels. Saturation density of this type of clone was 2- to 3-fold (1.7 to 2.1 X 10(5) cells/sq cm) higher than that of type II clones. Chromosomal analysis revealed that type I clones were human aneuploid ones with modal chromosome numbers ranging from 51 to 61. With the exception of clones 11 and 19, type I clones were able to produce tumors in athymic nude mice when injected s.c. Type II clones exhibited a more flattened and elongated appearance. The cells grew in a well-organized monolayer resembling fingerprint whorls. They showed lower saturation density (0.7 to 0.9 X 10(5) cells/sq cm). Chromosomal examinations revealed the clones to be human aneuploid ones with modal numbers from 47 to 54. Tumor formation was not observed in nude mice given injections of this type of cell. Both types of clones did not bear antigens cross-reacting with the antiserum against mouse spleen cells but had surface antigens which were affected by the antibody against HL-60 cells and complement. These results suggested that this human sarcoma was heterologous and that cells with widely different tumorigenic potential preexisted in the parental cell population.

Thrombospondin 2 gene expression is correlated with decreased vascularity in non-small cell lung cancer.

Stromal vascularity is thought to be a major factor involved in the progression of carcinoma. However, the crucial mechanisms of vascularization in the stroma are not well understood. Vascularity could be regulated by various cytokines produced by neoplastic or stromal cells in carcinoma. Thrombospondin (TSP) has an inhibitory role against vascularization in vitro, although the biological significance of TSP has not been characterized in vivo. We examined expression of TSP1 and TSP2 genes in 78 non-small cell lung cancers (NSCLCs) and 33 extraneoplastic lung tissue samples by reverse transcription-PCR. TSP1 expression was detected in 66.7% (52 of 78) of NSCLCs and in 69.7% (23 of 33) of extraneoplastic lung tissue specimens. TSP2 expression was seen in 48.7% (38 of 78) of NSCLCs, whereas 72.7% (24 of 33) of extraneoplastic lung tissue samples showed TSP2 gene expression. TSP2 expression was significantly decreased in NSCLC as compared with extraneoplastic lung tissue (chi2 test, P=0.019). Vascularity in the NSCLC was inversely correlated with TSP2 gene expression (Mann-Whitney U test, P=0.009). Patients with adenocarcinoma positive for TSP2 gene expression (22 of 49) showed significantly better prognosis than those without TSP2 (27 of 49; Cox-Mantel test, P=0.034). TSP1 expression showed no apparent correlation with these factors. These results suggested that TSP2 had an inhibitory role against vascularization and progression of NSCLC.

Electron microscopic identification of the intracellular secretion pathway of human G-CSF in a human tumor cell line: a comparative study with a Chinese hamster ovary cell line (IA1-7) transfected with human G-CSF cDNA.

Using monoclonal antibodies specific for human granulocyte colony-stimulating factor (G-CSF), intracellular localization of G-CSF in a G-CSF-producing human tumor cell line (CHU-2) and its ultrastructural characters were described and compared with those of a Chinese hamster ovary cell line (IA1-7) transfected with human G-CSF cDNA. The CHU-2 line, which was derived from a poorly differentiated squamous cell carcinoma of the oral cavity, preserved the character of a poorly differentiated squamous cell carcinoma. In the CHU-2 cell line, there were few cells immunohistochemically positive for G-CSF under light microscopic analysis despite the high transcription level of G-CSF cDNA and secretion of G-CSF that were comparable with cDNA-transfected IA1-7 cells. Using electron microscopy, the reaction products were localized mainly in the perinuclear space (PNS) and rough endoplasmic reticula (RER) without dilation of the cisternae, but they were very rarely found in the Golgi complex and not at all in other intracellular organelles. In contrast, most cells were positive for G-CSF in the IA1-7 cell line. Reaction products in this cell line were also demonstrated in the PNS and RER without dilation of the cisternae. These immunohistochemical findings, in conjunction with the results of Western and Northern blot analysis, suggested that G-CSF was secreted via the PNS and RER without intracellular retention.

Suppression of serum 1,25-dihydroxyvitamin D in humoral hypercalcemia of malignancy is caused by elaboration of a factor that inhibits renal 1,25-dihydroxyvitamin D3 production.

Although many patients with humoral hypercalcemia of malignancy exhibit reduced serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels, N-terminal fragments of recently identified PTH-related protein as well as PTH itself elevate serum 1,25-(OH)2D concentrations. In the present study, the effect of tumor extracts from human tumor-implanted hypercalcemic nude rat models with high and low serum 1,25-(OH)2D on renal 1,25-(OH)2D3 production was examined using rat kidney cells in culture. Whereas tumors from rats with high serum 1,25-(OH)2D levels (OCC rats) contained only a single peak of cAMP production-stimulating activity (CPSA) in osteogenic sarcoma cells on reverse phase HPLC, tumor extracts from rats with low serum 1,25-(OH)2D levels (UCC rats) contained at least two peaks of CPSA. The main peak (peak A) was estimated to be approximately 17K by gel permeation chromatography, which was the same as the molecular size of the hitherto identified PTH-related protein, and a minor peak of CPSA (peak B) was estimated to be about 25K. When peak A or crude extracts of OCC tumors as well as human PTH-(1-34) were added to primary cultures of rat kidney cells, the production of 1,25-(OH)2D3 was significantly stimulated. In contrast, although peak B or crude UCC tumor extracts had no effect on 1,25-(OH)2D3 production in themselves, when they were added together with peak A or human PTH-(1-34) the stimulation of 1,25-(OH)2D3 production was almost completely inhibited. Both peak A and peak B enhanced cAMP production in cultured kidney cells, and the cAMP production by peak A was not affected by peak B. These results are consistent with the possibility that elaboration of an additional factor from tumor cells may be the mechanism by which serum 1,25-(OH)2D levels are suppressed in patients with humoral hypercalcemia of malignancy. The nature as well as the mechanism of action of this factor remain to be elucidated.

Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma.

Human pancreatic cancer is a lethal malignancy, and the lesions show a very high incidence of point mutations of the K-ras oncogene. These alterations can be used as potential targets for specific ribozyme (Rz)-mediated growth suppression of the cancer cells. We designed an anti-K-ras Rz against mutant K-ras gene transcripts (codon 12, GGT to GTT) and generated a recombinant adenovirus (rAd) to express the Rz (rAd/anti-K-ras Rz). More than 95% of Capan-1 human pancreatic cells were infected with rAd/anti-K-ras Rz when treated with the virus at 200 plaque-forming units/cell. The virus, rAd/anti-K-ras Rz, significantly suppressed mutant K-ras gene expression and inhibited the growth of Capan-1 cells. At 3 days postinfection, we observed maximum growth suppression of the cells, characteristic morphological changes of apoptosis such as nuclear condensation and oligonucleosomal DNA fragmentation, and suppression of bcl-2 oncoprotein. These changes were not found in control virus-infected cells. Our results indicated that the virus rAd/anti-K-ras Rz specifically down-regulated the K-ras/bcl-2 pathway and induced apoptotic changes in Capan-1 pancreatic carcinoma cells. High-efficiency adenovirus-mediated delivery of anti-K-ras Rz could become a significant gene therapy strategy against human pancreatic cancer.

Ribozyme approach to downregulate vascular endothelial growth factor (VEGF) 189 expression in non-small cell lung cancer (NSCLC).

The aim of this study was to further clarify the role of the cell-associated isoform of vascular endothelial growth factor (VEGF189) on tumour growth and vascularity. Five isoforms of VEGF have been identified with different biological activities. VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 are generated by alternative splicing. We used a hammerhead-type ribozyme (V189Rz) to suppress VEGF189 mRNA. The V189Rz specifically cleaved exon 6 of VEGF189 mRNA, but showed no activity against the VEGF121 or VEGF165 isoforms. The V189Rz was introduced into the human non-small cell lung cancer (NSCLC) cell line (OZ-6/VR). The expression level of VEGF189 mRNA was decreased in the OZ-6/VR cells, while VEGF121 and 165 expression was unaltered. The OZ-6/VR cells xenotransplanted into nude mice showed markedly reduced vascularisation and growth, whereas the cell line did not show any decreased growth under tissue culture conditions. The OZ-6/VR cells (1 x 10(5) cells/mouse) formed no tumours, whereas the parental OZ-6 cells formed large tumours within 8 weeks. The specific suppression of VEGF189 by the ribozyme decreased vascularity and xenotransplantability of the lung cancer cell line. Thus, the cell-associated isoform of VEGF, VEGF189, might have a key role in stromal vascularisation and the growth of NSCLC xenografts in vivo.

Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma.

Vascular endothelial growth factor (VEGF) has five isoforms (VEGF206, 189, 165, 145 and 121). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF121 alone, 10 showed VEGF121 + 165, and 33 showed the VEGF121 + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF121 + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF121 + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the growth of RCC.

Growth stimulation of non-small cell lung cancer xenografts by granulocyte-macrophage colony-stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been suggested to be involved in the carcinogenesis of some types of tumours by autocrine or paracrine mechanisms. We examined GM-CSF/GM-CSF receptor (GM-CSFR) gene expression in 20 human non-small cell lung cancer (NSCLC) xenografts. The stimulatory effects of GM-CSF were examined using GM-CSF transgenic severe combined immunodeficient (SCID) mice (GM-Tg-SCID), which produce abundant human GM-CSF. A NSCLC xenograft (LC11-JCK), expressed GM-CSFR but not GM-CSF, and showed more rapid growth in GM-Tg-SCID than non-GM-CSF transgenic SCID mice (non-Tg-SCID). GM-CSF gene expression was detected in 48 of 90 (53%) primary NSCLC human specimens and GM-CSFR gene expression was detected in 42 specimens (47%). GM-CSF expression was detected in 13 of 30 squamous cell carcinoma specimens (43%) and GM-CSFR expression was detected in 10 specimens (33%). Patients with squamous cell carcinoma coexpressing GM-CSF and GM-CSFR showed significantly poorer prognosis than those expressing neither GM-CSF nor GM-CSFR (P < 0.05, Cox-Mantel test). These results suggest that GM-CSF can have a stimulatory effect on some NSCLC.

Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer.

Multidrug resistance-associated protein (MRP) is one of the major factors responsible for non-P-glycoprotein (Pgp)-mediated multidrug resistance of human tumour cells. In this study, we examined MRP and aberrant p53 expression in 54 colorectal cancers (CRC), 35 carcinoma in adenomas (CIA) and 40 adenomatous polyps by immunohistochemical procedures. 38 of 54 (70%) CRCs, 16 of 35 (46%) CIAs and 3 of 40 (8%) adenomatous polyps were MRP positive (chi 2 test, P < 0.0001). 36/54 (67%) CRCs, 10/35 (29%) CIAs and 0/40 adenomatous polyps were p53 positive. 30 of the 36 p53-positive CRCs were also MRP positive and 8/10 CIAs were both p53 and MRP positive. MRP overexpression correlated with aberrant p53 accumulation in CRCs and CIAs (chi 2 test, P < = or 0.01). Coexpression of MRP and p53 in the same cells was confirmed in the CRCs and CIAs by double staining procedures. These results suggested that MRP overexpression is related to aberrant p53 expression in CRC.

Cell-retained isoforms of vascular endothelial growth factor (VEGF) are correlated with poor prognosis in osteosarcoma.

Vascular endothelial growth factor (VEGF) is a major angiogenic factor. Osteosarcoma is characterised by hypervascularity and metastatic potential. We examined VEGF mRNA expression, VEGF isoform pattern and VEGF receptor (flt-1 and KDR) by RT-PCR analysis in 30 osteosarcomas. All 30 osteosarcomas expressed VEGF mRNA. 17 osteosarcomas (57%) expressed flt-1 mRNA, whilst 20 (67%) expressed KDR mRNA. 6/30 (20%) osteosarcomas were positive for VEGF121 only, 8 (27%) for VEGF121 + VEGF165, and 16 (53%) for VEGF121 + VEGF165 + VEGF189. Patients with osteosarcomas with VEGF165 (n = 24) had significantly poorer prognosis in comparison with those without VEGF165 (P = 0.022, Wilcoxon's test). The osteosarcomas with VEGF165 had significantly increased vascularity assessed on sections immunostained for CD34 (P < 0.001, Mann-Whitney U test). Although VEGF165 is a soluble isoform, it is also retained on the cellular surface. These results suggest that cell-retained VEGF isoforms (VEGF165, VEGF189) might be essential for neovascularisation in osteosarcoma, whilst the soluble VEGF121 isoform is not sufficient to stimulate neovascularisation in this type of neoplasm.

Polypoid carcinoma of the esophagus. A unifying term for "carcinosarcoma" and "pseudosarcoma".

Polypoid carcinomas with spindle-cell sarcomatous features have been designated either as carcinosarcoma or pseudosarcoma. The distinction between these two tumors has depended on the presence of "intermingling" of the carcinomatous and sarcomatous components in so-called carcinosarcoma. But unlike the carcinosarcoma, the sarcomatous component in pseudosarcoma has been considered benign. A polypoid tumor of the esophagus in a 57-year-old male was predominantly composed of spindle cell, sarcomatous cells. The presence of adjacent intraepithelial epidermoid carcinoma with transitional and ultrastructural features confirmed the epithelial origin of this tumor. Because of the absence of "intermingling," the primary tumor was considered to be a pseudosarcoma. However, our postmortem examination showed metastases composed of both carcinomatous and sarcomatous features. A review of the literature on carcinosarcomas and pseudosarcomas shows that only one case of pseudosarcoma reported by Hughes and Cruickshank showed a similar situation and indicates that the sarcomatous component in pseudosarcomas has the same metastatic potentiality as has been reported in carcinosarcomas. We conclude from these studies a basic similarity of the carcinosarcoma and pseudosarcoma. The term polypoid carcinoma is proposed for both these lesions.