Examination of percutaneous application in a 26-week carcinogenicity test in CB6F1-TG rasH2 mice.

We examined the possibility of expanding applications of rasH2 mice, which are genetically manipulated mice for short-term carcinogenicity tests, to percutaneous application. A 26-week short-term carcinogenicity study was performed on a total of 300 mice including 75 male and female rasH2 mice each, and 75 male and female non-Tg mice each from the same litter as the rasH2 mice divided into untreated group, an ethanol group, a white Vaseline group, an acetone group, and a phorbol 12-myristate 13-acetate (TPA) group. Only shaving of dorsal skin was performed on the untreated mice. As a positive control, TPA was administered percutaneously at a dose of 2.5 microg/kg and 3 times/week for 26 weeks based on the protocol for Tg.AC mice in the ILSI/HESI international validation study. In the ethanol, white Vaseline, and acetone groups, no tumorous changes were observed on the skin at the administration site. In the TPA group, nodular changes at the administration site were observed from seven weeks after the start of administration in rasH2 mice, and the incidence in males and females was 50.0% (7/14) and 53.3% (8/15), respectively. In a pathological examination, nodules in 21.4% (3/14) of males and 46.7% (7/15) of females were diagnosed as skin papilloma or keratoacanthoma, and the rest as squamous cell hyperplasia. In the non-Tg mice, no nodules or tumorigenic changes were observed at the administration site. These findings show that percutaneous application in rasH2 mice is possible in 26-week carcinogenicity tests.

Use of IC tags in short-term carcinogenicity study on CB6F1 TGrasH2 mice.

We studied the effect of IC tags, subcutaneously implanted animal identification tools, on rasH2 mice. A 26-week short-term carcinogenicity study was performed on a total of 299 mice including 75 male and female rasH2 mice each, and 74 male and 75 female non-Tg mice from the same litter as the rasH2 mice divided into a non-IC tag group, the IC-tag group, acetone group, TPA group and MNU group (all of the animals except for those in the non-IC tag group) had IC tags implanted subcutaneously in their backs. The administration methods of the positive control drugs TPA (2.5 micro g/kg, 3 times/week, percutaneously) and MNU (75 mg/kg, single intraperitoneal injection) were based on the protocol of the ILSI/HESI international collaborative study. The results showed no differences in the tumorigenic incidence and organs developing tumors between the IC tag and non-IC tag groups in both rasH2 and non-Tg mice. In the positive control MNU group, the tumorigenic incidence and organs developing tumors were the same as the background data and no promotion of carcinogenesis was observed. In all IC tag groups including the TPA group and MNU group, a fibrous capsule was formed around the IC tags subcutaneously, but no inflammatory changes or neoplastic changes were observed. From these findings, it was concluded that the IC tag has no effect on a 26-week carcinogenicity test of rasH2 mice under the conditions of the present study.

Unique properties of 189 amino acid isoform of vascular endothelial growth factor in tumorigenesis.

The 189 amino acid isoform of vascular endothelial growth factor (VEGF189) has been shown to be more strongly associated with the cell membrane than other isoforms of human VEGF (VEGF121, VEGF165). To analyze the biological activities of these VEGF isoforms on tumor growth, we transfected human VEGF121, VEGF165 or VEGF189 cDNA into the human colon cancer cell line SW-480, and established several clones overexpressing these VEGF isoforms. The total amounts of VEGF protein in the culture supernatants of the VEGF189-transfectants were less than those of VEGF121 and VEGF165-transfectants. These transfectants showed no significant differences in growth in culture. Nevertheless, the rate of in vivo tumor growth of VEGF189-transfectants was faster than or equivalent to that of VEGF121-transfectants, while the VEGF165-transfectant showed the greatest enhancement of tumor growth. The protein levels of VEGF were markedly increased only in the VEGF189-transfectants cultured in the presence of heparin. The enhanced in vivo tumor growth of VEGF189-transfectants can be partly explained by the cell-associated features of VEGF189 molecules. The VEGF189 molecule, which is strongly bound to the cell surface, has unique properties and high potential in local angiogenesis and tumor growth in the cancer inductive microenvironment.

Human thrombospondin 2 inhibits proliferation of microvascular endothelial cells.

This study was performed to characterize human thrombospondin 2 (TSP2). TSP2 has recently attracted attention as an endogenous negative regulator of angiogenesis in tumorigenesis. We cloned and transfected human TSP2 cDNA into the human colon cancer cell line SW-480. Stable transfectants (TSP2-1, TSP2-6) overexpressing TSP2 were established. Growth characteristics of TSP2-transfectants were investigated in vitro and in vivo. TSP2-transfectants showed similar growth properties to vector-transfectants and wild-type SW-480 cells. The overexpression of transfected human TSP2 cDNA did not affect proliferation of SW-480 cells. When the conditioned media of TSP2-transfectants were added to cultures of bovine pulmonary microvascular endothelial cells (BPMEC), the BPMEC proliferation was significantly inhibited. These results suggested that human TSP2 is a potential inhibitor of angiogenesis.