Quivo Tahin

Carcinogenicity of estrogens in human breast epithelial cells.

Epidemiological and clinical evidences indicate that breast cancer risk is associated with prolonged ovarian function that results in elevated circulating levels of steroid hormones. Principal among these is estrogen, which is associated with two important risk factors, early onset of menarche and late menopause. However, up to now there is no direct experimental evidence that estrogens are responsible of the initiation of human breast cancer. We postulate that if estrogens are causative agents of this disease, they should elicit in human breast epithelial cells (HBEC) genomic alterations similar to those exhibited by human breast cancers, such as DNA amplification and loss of genetic material representing tumor suppressor genes. These effects could result from binding of the hormone to its nuclear receptors (ER) or from its metabolic activation to reactive metabolites. This hypothesis was tested by treating with the natural estrogen 17β‐estradiol (E2) and the synthetic steroid diethylstilbestrol (DES) MCF‐10F cells, a HBEC line that is negative for ER. Cells treated with the chemical carcinogen benzo (a) pyrene (BP) served as a positive control of cell transformation. BP‐, E2‐, and DES‐treated MCF‐10F cells showed increases in survival efficiency and colony efficiency in agar methocel, and loss of ductulogenic capacity in collagen gel. The largest colonies were formed by BP‐treated cells, becoming progressively smaller in DES‐ and E2‐treated cells. The loss of ductulogenic capacity was maximal in BP‐, and less prominent in E2‐ and DES‐treated cells. Genomic analysis revealed that E2‐ and DES‐treated cells exhibited loss of heterozygosity in chromosomes 3 and 11, at 3p21, 3p21–21.2, 3p21.1–14.2, and 3p14.2–14.1, and at 11q23.3 and 11q23.1–25 regions, respectively. It is noteworthy that these loci are also affected in breast lesions, such as ductal hyperplasia, carcinoma in situ, and invasive carcinoma. Our data are the first ones to demonstrate that estrogens induce in HBEC phenotypic changes indicative of cell transformation and that those changes are associated with significant genomic alterations that might unravel new pathways in the initiation of breast cancer.

Hormone receptors and cathepsin D levels in human breast epithelial cells transformed by chemical carcinogens and c-Ha-ras transfection.

SummaryThe objective of this work was to determine whether transformation of the human breast epithelial cell line MCF-10F by the chemical carcinogens 7, 12-dimethylbenz(a)anthracene (DMBA) or benzo(a)pyrene (BP), or c-Ha-ras oncogene transfection, influence the expression of epidermal growth factor receptor (EGFR), estrogen (ER) or progesterone (PR) receptors, and the content of cathepsin-D (Cath.D). MCF-10F control cells did not express any of the phenotypes of neoplastic transformation, whereas carcinogen-treated cells and clones derived from the latter formed colonies in agar-methocel, and exhibited increased chemotaxis and chemoinvasion. Clone BP-1E was also tumorigenic in SCID mice. The BP1 cell line transfected with mutated c-Ha-ras oncogene, named BP1-Tras, became more aggressive after transfection and decreased the latency time to tumorigenesis. Radioligand binding and immunocytochemical reactions were utilized for determining the receptors and Cath.D content of control and carcinogen-treated cells and their derived clones. MCF-10F cells contained 37 fmol/mg of protein of EGFR, ER and PR were undetectable, and Cath.D content was 70 fmol/mg protein. EGFR content was significantly higher in D3-1 and BP1-E cell lines vs the control MCF-10F and the other DMBA and BP clones, correlating positively with the emergence of the transformation phenotype. Whereas EGFR levels were not significantly different in BP1-Tras cells when compared with BP1-E, the former were more tumorigenic in SCID mice, an observation suggesting an alternative pathway in these cells in the formation of tumors. PR, ER, and Cath.D content was not modified by either carcinogen treatment or ras transfection in most of the clones and subclones, except clone BP2 that has a significant increase in ER content, and was not associated with any of the neoplastic phenotypes. These data allowed us to conclude that the level of EGFR is associated with the expression of carcinogen-induced transformation phenotypes whereas ER, PR, and Cath.D did not seem to be modified during the process of chemically induced or c-Ha-ras enhanced transformation of MCF-10F cells.

Chromosome 17p13.2 transfer reverts transformation phenotypes and fas-mediated apoptosis in breast epithelial cells

Transformation of the human breast epithelial cells (HBEC) MCF‐10F with the carcinogen benz(a)pyrene (BP) into BP1‐E cells resulted in the loss of the chromosome 17 p13.2 locus (D17S796 marker) and formation of colonies in agar‐methocel (colony efficiency (CE)), loss of ductulogenic capacity in collagen matrix, and resistance to anti‐Fas monoclonal antibody (Mab)‐induced apoptosis. For testing the role of that specific region of chromosome 17 in the expression of transformation phenotypes, we transferred chromosome 17 from mouse fibroblast donors to BP1‐E cells. Chromosome 11 was used as negative control. After G418 selection, nine clones each were randomly selected from BP1‐E‐11neo and BP1‐E‐17neo hybrids, respectively, and tested for the presence of the donor chromosomes by fluorescent in situ hybridization and polymerase chain reaction‐based restriction fragment length polymorphism (PCR‐RFLP) analyses. Sensitivity to Fas Mab–induced apoptosis and evaluation of transformation phenotype expression were tested in MCF‐10F, BP1‐E, and nine BP1‐E‐11neo and BP1‐E‐17neo clones each. Six BP1‐E‐17neo clones exhibited a reversion of transformation phenotypes and a dose dependent sensitivity to Fas Mab‐induced apoptosis, behaving similarly to MCF‐10F cells. All BP1‐E‐11neo, and three BP1‐E‐17neo cell clones, like BP1‐E cells, retained a high CE, loss of ductulogenic capacity, and were resistant to all Fas Mab doses tested. Genomic analysis revealed that those six BP1‐E‐17neo clones that were Fas‐sensitive and reverted their transformed phenotypes had retained the 17p13.2 (D17S796 marker) region, whereas it was absent in all resistant clones, indicating that the expression of transformation phenotypes and the sensitivity of the cells to Fas‐mediated apoptosis were under the control of genes located in this region.

ROLE OF RAS ONCOGENE IN HUMAN BREAST CANCER: AN EXPERIMENTAL APPROACH

There are several reports indicating that oncogenes are associated with breast cancer in humans and animals (1,2). Among the oncogenes, the ras family with their three variants, c-Ha-ras, v-Ha-ras and n-Ha-ras, has been widely studied and reported to be involved in mammary carcinomas induced in experimental animals by chemical carcinogens, in breast cancer cell lines, in primary breast cancers, as rare alleles or as an amplification or expression of the gene product of the ras gene, the p21 protein (3 – 6). However, whether the ras oncogene is a causative agent of breast cancer has not been proven as yet.