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Subtractive hybridization was used to isolate genes expressed uniquely in the immortalized human breast epithelial cell (HBEC) line MCF-10F and not in the mortal HBEC line S-130, from which MCF-10F cells were derived. We identified a 233-bp cDNA that was expressed in MCF-10F cells and not in their mortal counterpart S-130 cells. Sequence comparison with the GenBank database revealed that the cDNA was identical to the gene encoding human ferritin heavy H chain. Northern blot analysis using the isolated cDNA as a probe showed a differentially expressed 1.1-kb transcript of ferritin H in total RNA from the immortal MCF-10F cells, MCF-10F cells treated with the chemical carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene, and the breast cancer cell lines MCF-7, HBL-100, T-47D, and BT-20. No ferritin H transcript was detected in the mortal line S-130 or in other primary HBEC cultures. Increased levels of mRNA transcript signals were also detected in total RNA from breast cancer tissue samples. Tissue with ductal hyperplasia had higher expression levels than normal adjacent mammary tissue. In situ hybridization showed high levels of ferritin H transcript in mammary tissue areas with ductal hyperplasia, carcinoma in situ, and infiltrating ductal carcinoma. This is the first report of the differential expression and upregulation of human ferritin H chain gene in immortal HBECs. It may be an important factor in the process of immortalization, possibly an early stage of malignant transformation of HBECs, providing cells with iron necessary for growth and clonal expansion. Also, ferritin iron, once released, may increase the level of reactive iron, leading to an increase in oxygen free-radical generation, oxidative DNA damage, and mutation. Mol. Carcinog. 20:332–339, 1997. © 1997 Wiley-Liss, Inc.

In the present study we have examined the effect of dexamethasone on ob gene mRNA expression and leptin release from isolated human subcutaneous adipocytes. Dexamethasone stimulated leptin release from cultured adipocytes in a time- and dose-dependent manner. A two-fold increase in leptin release was detectable by 36 h of treatment with 10(-7) M dexamethasone. Leptin release was preceded by a significant 83 +/- 30% increase in ob mRNA after 24 h exposure to the compound. Co-incubation of cells with dexamethasone (10(-7) M) and insulin (10(-7) or 10(-9) M) completely blocked the dexamethasone-stimulated increase in ob mRNA and leptin release. These data demonstrate that insulin and glucocorticoids regulate leptin synthesis and release from human adipocytes in vitro.

In order to determine what is the role of genomic alterations in neoplasia, it is necessary to verify what is their function at each one of the various stages of breast cancer progression, which initiates as ductal hyperplasia, progressing to atypical ductal hyperplasia, carcinoma in situ, and invasive and metastatic adenocarcinoma. These studies are hindered by the fact that these stages are not always present in a single patient, or in a sequential manner, or when present the available material might be insufficient or inadequate for performing genetic manipulation for testing their functional role. These disadvantages have been overcome by utilizing an in vitro model of cell transformation, the spontaneously immortalized human breast epithelial cell line (HBEC) MCF- 10F, which upon treatment with the chemical carcinogens benzo(a)pyrene (BP) or 7,12-dimethyl-benz(a)anthracene (DMBA) expresses in a sequential fashion all the phenotypes indicative of neoplastic transformation, i.e., increased survival in agar, formation of colonies in agar methocel, invasiveness in a matrigel in vitro system, and tumorigenesis in severe combined immunodeficient (SCID) mice. The immortalization of MCF-1 OF cells is associated with the presence of a point mutation in exon 7 of the tumor suppressor gene p53. Carcinogen-induced transformation was manifested as colony formation in agar methocel and anchorage independence, which were associated with loss of heterozygosity (LOH) and point mutations in codons 12 and 61 of c-Ha-ras gene. Other oncogenes involved in this early stage were c-neu, c-myc, and int-2. The expression of the tumorigenic phenotype was associated with LOH in the telomeric portion of chromosome 17 and overexpression of the mdm2 gene. This in vitro system provides all of the sequential steps to neoplastic transformation in a reproducible manner, thus providing a paradigm for clarifying the role of the genetic alterations in tumor initiation and progression. © 1995 Wiley-Liss, Inc.