No abstract

Sporadic breast cancer, the most common cancer diagnosed in American and Northern European women, is gradually increasing in incidence in most Western countries. Prevention would be the most efficient way of eradicating this disease. This goal, however, cannot be accomplished until the specific agent(s) or mechanisms that initiate the neoplastic process are identified. Experimental studies have demonstrated that mammary cancer is a hormone-dependent multistep process that can be induced by a variety of compounds and mechanisms, that is, hormones, chemicals, radiation, and viruses, in addition to or in combination with genetic factors. Although estrogens have been shown to play a central role in breast cancer development, their carcinogenicity on human breast epithelial cells (HBECs) has not yet been clearly demonstrated. Breast cancer initiates in the undifferentiated lobules type 1, which are composed of three cell types: highly proliferating cells that are estrogen-receptor negative (ERϪ), nonproliferating cells that are ER positive (ERϩ), and very few (Ͻ1%) ERϩ cells that proliferate. Interestingly, endogenous 17␤-estradiol (E 2 ) is metabolized by the cytochrome P450 enzyme isoforms CYP1A1 and CYP1B1, which also activate benzo[a]pyrene (B[a]P), a carcinogen contained in cigarette smoke. We postulate that if estrogens are carcinogenic in HBECs, they should induce the same transformation phenotypes induced by chemical carcinogens and ultimately genomic changes observed in spontaneously developing primary breast cancers. To test this hypothesis we compared the transforming potential of E 2 on the HBEC MCF-10F with that of B[a]P. Both E 2 and B[a]P induced anchorage-independent growth, colony formation in agar methocel, and loss of ductulogenic capacity in collagen gel, all parameters indicative of cell transformation. In addition, the DNA of E 2 -transformed cells expressed LOH in chromosome 11 at 11q23.3, 11q24.2-q25, and LOH at 13q12-q13. B[a]P-induced cell transformation was also associated with LOH at 13q12-q13 and at 17p13.2. The relevance of these findings is highlighted by the observation that E 2 -and B[a]P-induced genomic alterations in the same loci found in ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma of the breast. Environ. Mol. Mutagen. 39:254-263, 2002.

Based upon our previous observations that the susceptibility of the mammary gland to neoplastic transformation is related to its degree of development and proliferative activity,8-10 we studied 15 reduction mammoplasties by determining the degree of breast development, measured based upon the type and number of lobules present," which allowed us to classify the mammoplasty specimens into two types: (a) poorly differentiated breasts, those composed oftype 1 and 2 lobules; and (b) well-differentiated breasts, those composed almost exclusively of type 3 lobules.1° Portions of the same breasts were utilized for measuring the rate of DNA synthesis or DNA labeling index (DNA-LI) by in vim incorporation of 3H-thymidine and for studying the in vim growth characteristics of organoids obtained by digestion of the tissue prior to plating. It was found that 10 of the breasts were composed predominantly of type 1 and 2 lobules, and their DNA-LI was 1.03 f 0.48. Cells derived from these samples attached to the culture dish immediately, with a high number of doublings (0.64 f 0.47). Five samples were composed almost exclusively of type 3 lobules. Their DNA-LI was 0.05 f 0.05, markedly lower than the DNA-LI of cells derived fiom poorly differentiated breasts; the dfferences were statistically significant (FIG. 1). The cells' number of doublings was 0.11 f 0.06. There was a high correlation coefficient (r = 0.802) between the number of doublings of cells in primary cultures and the percentage of type 1 and 2 lobules present in the specimen, whereas differentiation inversely correlated with this parameter (r = 0.722) (FIGS. 2 and 3). 1.6 1 --If DNA-LI FIGURE 1. Influence of lobulardevelopment on the number of doublingdday and DNAlabeling index. RUSSO ct uf.: CRITICAL STEPS 1 -0 -3 Y=0.0*2 -0 . 2 6 0~ @ \ @ I 1 0 Y=O. 126+0.35Bx Corr. =O .802 FIGURE 2. Correlation of doublingdday and type 1 and type 2 lobular structures. RESPONSE OF HUMAN BREAST EPITHELIAL CELLS IN PRIMARY CULTURES To CARCINOGEN TREATMENTTwenty additional reduction mammoplasty specimens were digested for obtention of organoids, which were classified as type 1, 2, or 3 lobules. The organoids were plated in DMEM:F12 culture medium containing 1.05 mM calcium (Ca++). When they formed monolayers, the cells were passaged and then treated with the following carcinogens: 7,12-dimethylbenz(a)anthracene (DMBA), N-methyl-N-nitrosourea (NMU), methyl-N-nitro-nitrosoguanidine

2. Introduction. 3. Human Breast Epithelial Cells (HBEC) in Culture 4. Factors Influencing Susceptibility of HBEC to Cell Transformation 4.1. Lobular differentiation 4.2. Genetic predisposition 4.3. Cell immortalization 5. Molecular Mechanisms of Cell Immortalization 5.1. Activation of telomerase 5.2. Abrogation of cell cycle control 5.3. Genes preferentially expressed during cell immortalization 6. Molecular Mechanisms of Cell Transformation 6.1. Epigenetic mechanisms 6.2. Genetic mechanisms 7. Genomic changes in Immortalization and Transformation of HBEC 7.1. Genomic changes in cell immortalization 7.2. Genomic changes in cell transformation 8. Genomic Changes in Human Breast Lesions 9. Functional Roles of Chromosomes 11 and 17 in Transformed Phenotype Expression of HBEC 10. Summary and Perspectives 11. Acknowledgments 12. References

The influence of diets having different fatty acids composition on the fatty acid content of (the phospholipids) of rat liver mitochondria and microsomes, heart mitochondria, brain mitochondria and microsonies has been analyzed. It has been found that each organelle has its own peculiar composition in fatty acids. This composition may be profoundly influenced by the diet, but to different degrees in different organelles. Those of brain are most resistant. The changes observed are rather rapid, being generally already maximal after three weeks of treatment. The parallel between fatty acid composition of diets, and the changes observed in the organelles, is not strict, and is probably influenced by the metabolic competition among oleic acid, linoleic acid, linolenic acid. Unusual fatty acids like crucic acid. trans‐oleic acid. and trans‐linoleic acid can also become incorporated into the membranes of cell organelles.