64 Ma et al. Fig. 2. Immunostaining for SMA. A) Immunoperoxidase reaction with anti-alpha smooth muscle actin antibody reveals myoepithelial cells lining nests of secretory cells in intraductal papilloma. Nuclei counterstained with HE×100. B) Immunoperoxidase reaction with anti-alpha smooth muscle actin antibody reveals that myoepithelial cells scattered at the base of nests in fibroadenoma. Nuclei counter- stained with HE×100. C) Immunoperoxidase reaction with anti- alpha smooth muscle actin antibody reveals that myoepithelial cells scattered at the base of nests and stretched in adenocarcinoma. Nuclei counterstained with HE×100. papilloma (Fig. 2C). Fibroadenoma showed immunohisto- chemistry patterns which were intermediate between intra- ductal papilloma and adenocarcinoma (Fig. 2B). ERα positive cells were indicated by intensely stained nuclei and observed in all tumors (Fig. 3A–C). There were no significant differences in the appearance and the number of ERα positive cells among the three histological types of rat breast tumors. PCNA immunostaining was confined to nuclei of tumor cells, suggesting proliferating cells (Fig. 4A–C). We quantified the PCNA-labeled cells in 30 intraductal papil- lomas, 10 fibroadenomas, and 4 adenocarcinomas using ACT-1 Image Analysis System (Nikon, Japan). The results of PCNA labeling index are shown in Table 3. PCNA label- ing index was 24±8% in intraductal papilloma, 36±8% in fibroadenoma, and 56±10% in adenocarcinoma. It was sig- nificantly different in the PCNA labeling index among the three histological types of rat breast tumors (p<0.001). IV. Discussion Considerable epidemiological studies have shown a positive association between milk and breast cancer [24, 27]. Ganmaa and Sato correlated incidence rates for breast, ovarian, and corpus uteri cancers with food intake in 40 countries [5], and put forth the hypothesis that milk and dairy products have a significant effect on the development of these cancers. However, there were few animal experi- ments to verify this hypothesis. The present study clearly demonstrated that the tumor incidence in the milk group was significantly higher than that in the water group and comparable to that in the estrone sulfate group. The milk group developed more than one third as many tumors as the water group. These results, therefore, suggest that milk as well as estrone sulfate has a stimulatory effect on the development of DMBA-induced rat mammary tumors. Group I II III Total number of tumors 53 33 17 Table 2. Histological type of tumors IDP (%) 44 (83%) 24 (73%) 14 (82%) Histological type FA (%) 7 (13%) 7 (21%) 3 (18%) AC (%) 2 (4%) 2 (6%) 0 (0%) I, milk group; II, estrone sulfate group; III, water group; IDP, intraductal papilloma; FA, fibroadenoma; AC, adenocarcinoma.