Table 3. Results of immunohistochemistry staining for PCNA Ma et al. No. of tumors 30 10 4 Mean labeling index±SD (%) 24±8 36±8 56±10 p p=0.003 p<0.001 IDP, intraductal papilloma; FA, fibroadenoma; AC, adenocarci- noma; SD, standard deviation. Some animal studies for breast cancer proved that high fat diets increase the incidence of spontaneous, chemically induced, and radiation-induced mammary tumors [1, 7]. Therefore, a high consumption of milk may results in a high dietary fat intake, particularly saturated fat, which in turn increases breast cancer risk. Another hypothesis suggesting an increased risk of breast cancer with high milk intake focuses on the insulin- like growth factor I (IGF-I). Some investigators found that bovine growth hormone, which is sometimes administered to dairy cattle to increase milk production, resulted in in- creased concentrations of IGF-I in milk [16]. IGF-I concen- trations in commercial milk have been reported to range from 6 to 162 ng/ml [13]. In a human study, plasma IGF-I concentration increased by 10% when healthy subjects con- sumed commercial milk [8]. IGF-I has been shown to pro- mote breast cancer cell growth and some experiments found that removing or blocking IGF-I receptors from the cell membrane can abolish viral or cellular oncogene-induced malignant transformation [31]. Therefore, IGF-I in milk may promote the development of tumors induced by DMBA in rat. Estrogens are breast cancer promoters because of their physiological role in stimulating the mammary gland [28]. Our present experiment also demonstrated that estrone sulfate can stimulate the development of tumors induced by DMBA in rat. Moreover, all tumors induced by DMBA in the present experiment were positive for ERα. Russo et al. reported that mammary tumors in the rat are generally strongly hormone-dependent for both induction and growth [19]. Qin et al. measured the estrogen concentration of com- mercial milk and recently reported that the estrogen concen- tration in commercial milk was about 703.3 pg/ml [17]. Although the concentration of estrogen in milk is not very high, Ganmaa et al. have proved that commercial milk has a biologically significant hormonal effect and reported that the uterotrophic effect of commercial milk was almost compara- ble to that of 100 ng/ml estrone sulfate solution [6]. In this respect, estrogen in milk can be responsible for tumor pro- motion. In our present experiment, we classified the mammary tumors induced by DMBA into three histological types: intraductal papilloma, fibroadenoma, and adenocarcinoma. The rates of benign tumors (intraductal papilloma and fibro- adenoma) were almost the same among the three groups. 66 Histological types IDP FA AC However, malignant tumor (adenocarcinoma) was found only in milk and estrone sulfate groups. Van Zwieten has reported that the administration of estrogen results in an in- crease in the proportion of rats with malignant tumors, espe- cially cribriform carcinomas [29]. Results of our present ex- periment suggested that milk as well as estrone sulfate could be associated with the occurrence of adenocarcinoma. According to histological appearance, we classified four tumors as malignant. However, SMA immunostaining of these four adenocarcinomas also presented positive. Papotti et al. studied the cellular composition of rat mam- mary carcinoma by both light and electron microscopy [15]. They found that mammary myoepithelial cells were a con- stant component in mammary carcinoma. Evidence of the presence of myoepithelial cells in DMBA-induced rat mammary carcinoma has also been reported in some other studies [11]. Therefore, although the presence of myoepithe- lial cells is commonly regarded as a hallmark of benignity in humans, myoepithelial cells will present as a constant in rat mammary carcinomas. PCNA is an essential replication factor to initiate cell cycle progression [12]. The number of PCNA-labeled cells generally is higher in malignant tumors than in benign ones. In our present experiment, PCNA labeling index of adenocarcinoma was significantly higher than that of intraductal papilloma and fibroadenoma. Thus, PCNA immunostaining is useful for histological classi- fication of rat mammary tumors. In conclusion, milk as well as estrone sulfate promoted the development of DMBA-induced mammary tumors in rat and could be associated with the occurrence of adeno- carcinoma. Immunohistochemical staining for SMA and PCNA was a useful auxiliary for histological classification of tumors. V. Acknowledgments This research was supported in part by a grant from the National Natural Science Foundation of China (No. 30471448) to Dr. Pei-Yu Wang. VI. References 1. Ballard-Barbash, R. and Swanson, C. A. (2004) Body weight: es- timation of risk for breast and endometrial cancers. Am. J. Clin. Nutr. 63; 437S–441S. 2. Bussolati, G. (1980) Actin-rich (myoepithelial) cells in lobular carcinoma in situ of the breast. Virchows Arch. B Cell Pathol. 32; 165–176. 3. Cleary, M. P. and Maihle, N. J. (1997) The role of body mass in- dex in the relative risk of developing premenopausal versus post- menopausal breast cancer. Proc. Soc. Exp. Biol. Med. 216; 28–43. 4. Coleman, M. P., Esteve, J., Damiecki, P., Arslan, A. and Renard, H. (1993) Trends in cancer incidence and mortality. IARC Sci. Publ. 121; 1–806. 5. Ganmaa, D. and Sato, A. (2005) The possible role of female sex hormones in milk from pregnant cows in the development of breast, ovarian and corpus uteri cancers. Med. 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